Novel acetal oxolane lipids

Abstract

The present invention is in the field of drug delivery and particularly lipid nanoparticles (LNP) as the preferred mode of delivering drug products in vivo. It generally relates to novel acetal oxolane lipids that can be used alone or in combination with other lipid components, such as cationic lipids, neutral lipids, cholesterol, PEG and polymer conjugated lipids, to form lipid nanoparticles with oligonucleotides, to facilitate the intracellular delivery of therapeutic nucleic acids (e.g. oligonucleotides, messenger RNA, Silencing RNA, small interfering RNAs (siRNAs), short hairpin RNA (shRNA)microRNAs (miRNAs) and small PIWI-interacting RNAs (piRNAs), Long non-coding RNAs (lncRNAs)) both in vitro and in vivo.

Description

[0001] NOVEL ACETAL OXOLANE LIPIDS

[0002] FIELD OF THE INVENTION

[0003] The present disclosure is in the field of drug delivery and particularly lipid nanoparticles (LNP) as the preferred mode of delivering drug products in vivo.

[0004] The present disclosure generally relates to novel acetal oxolane lipids that can be used alone or in combination with other lipid components, such as cationic lipids, neutral lipids, cholesterol, PEG and polymer conjugated lipids, to form lipid nanoparticles with oligonucleotides, to facilitate the intracellular delivery of therapeutic nucleic acids (e.g. oligonucleotides, messenger RNA, Silencing RNA, small interfering RNAs (siRNAs), short hairpin RNA (shRNA)microRNAs (miRNAs) and small PlWI-interacting RNAs (piRNAs), Long noncoding RNAs (IncRNAs)) both in vitro and in vivo.

[0005] BACKGROUND

[0006] Despite extensive research, there are challenges faced during the later stages of drug development typically due to safety and efficacy concerns that fundamentally arise from high accumulation in off-target organs or poor accumulation in target organs, respectively. This has been a major bottleneck in the translation of potent drug candidates, which inherently possess excellent potential but fail to demonstrate significant clinical impact due to dose-related toxi cities and / or dose-limited efficacies due to off-taiget effects.

[0007] Nanoparticles have been developed to target therapies to specific targets. It is understood that modulation of physicochemical properties such as size and charge could improve nanoparticles’ targeting to specific tissues (Maldonado, R. A., LaMothe, R. A., Ferrari, J. D., Zhang, A. H., Rossi, R. J., Kolte, P. N., Griset, A. P., O’Neil, C., Altreuter, D. H., Browning, E., et al. (2015). Proc. Natl. Acad. Sci. USA 112, E156–E165.). However, nanoparticles also face biological barriers that impede their targeting capabilities (Blanco, E., Shen, H., and Ferrari, M. (2015). Nat. Biotechnol. 33, 941-951).

[0008] Lipid nanoparticles (LNPs) then emerged across the pharmaceutical industry as promising vehicles to deliver a variety of therapeutic agents. Advanced therapeutic modalities are increasingly using lipid nanoparticles (LNP) as the preferred mode of delivering drug product. Lipids including ionizable lipids, cationic lipids and PEGylated lipids are components of LNPs which can be modified to guide the delivery of the LNPs to different tissues and cell types in the body.

[0009] LNPs have been successful in effectively protecting and transporting numerous therapeutic products such as small molecules, nucleic acids, peptides, antibodies, and cell-based strategies to cells. Thus, the application of Lipid nanoparticles (LNPs) has extended to other fields, such as medical imaging, cosmetics, nutrition, agriculture, and other innovative areas such as nanoreactors.

[0010] Infact the most recent breakthrough in the nanomedicine field was the development of LNP mRNA vaccines. Lipid NP delivery of unstable mRNA protects the mRNA molecule, thus allowing cell uptake and preventing immune responses. [ Reichmuth AM, Oberli MA, Jaklenec A, Langer R, Blankschtein D. mRNA vaccine delivery using lipid nanoparticles. Ther Deliv.

[0011] 2016; 7(5): 319-334]

[0012] However, lipid nanoparticles tend to naturally localize in the liver. The currently available entities lack high level selective tissue tropism resulting in lowering the efficiency in targeted delivery and bioavailability.

[0013] Cationic lipids are promising in mRNA delivery, however, they have shortcomings, such as instability in systemic circulation and immunogenicity. For example, intravenously administered cationic liposomes cause liver damage, exert strong interferon-y triggered inflammatory responses, and are neutralized by serum anionic opsonin proteins. [ Cherng JY, van de Wetering P, Talsma H, Crommelin DJ, Hennink WE. Effect of size and serum proteins on transfection efficiency of poly ((2-dimethylamino)ethyl methacrylate)-plasmid nanoparticles. Pharm Res. ].

[0014] Cationic lipids also offer great promise as carriers for the delivery of fragile compounds such as nucleic acids, some cationic lipids cause cytotoxicity. In some cases, cationic lipids reduce mitosis in cells, form vacuoles in the cytoplasms of cells, and cause detrimental effect on key cellular proteins such as protein kinase C. [Lv, H. T.; Zhang, S. B.; Wang, B.; Cui, S. H.; Yan, J. Toxicity of Cationic Lipids and Cationic Polymers in Gene Delivery. J. Controlled Release 2006, 114, 100-109.]. In many cases, the effectiveness of a cationic lipid in LNP formulations correlates to increased toxicity. For example, multivalent cationic lipids have been more effective than monovalent cationic lipids in LNP formulations but are also much more toxic to cells. [ Lipid Nanoparticles-From Liposomes to mRNA Vaccine Delivery, a Landscape of Research Diversity and Advancement; Rumiana Tenchov et.al; ACS Nano 2021, 15, 16982-17015]

[0015] Therefore, cationic lipids must be improved to increase stability, biocompatibility, and transfection efficiency. [Huotari J, Helenius A. Endosome maturation. EMBO J.

[0016] 2011;30(17):3481-3500. doi: 10.1038 / emboj.2011.286.].

[0017] The efficacy of LNPs depends on the optimal ratio of various components, including ionizable and cationic lipids, helper lipids, cholesterol, and PEG constituents, each playing crucial roles in cellular interactions, payload delivery, and overall nanoparticle stability. [Cullis & Hope, Mol Ther 2017 Jul 5;25(7):1467-1475].

[0018] Autosomal Dominant Polycystic Kidney Disease (ADPKD) is a highly prevalent genetic disease often characterized by the progressive development of fluid-filled cysts in both kidneys causing their gradual enlargement and ultimately leading to End-Stage Kidney Disease (ESKD)l overtime. This debilitating disease affects almost 12.5 million worldwide and is the leading cause of ESKD2. Almost 85% of global incidences of ADPKD involved mutations in the PKD1 gene on chromosome 16 whereas the rest 15% reported mutations in the PKD2 gene on chromosome. These genes code for essential proteins such as poly cystin 1 and polycystin 2 which are key regulators of cAMP, mTOR and epidermal growth factor signalling pathways. In addition to renal complications, ADPKD is also associated with other extrarenal manifestations such as liver and pancreatic cysts, cardiac abnormalities, and an increased risk of intracranial aneurisms. Existing strategies for the treatment of ADPKD primarily focus on symptomatic management and slowing disease progression instead of treating its root cause. The only FDA approved drug, Tolvaptan, focuses on inhibiting vasopressin V2 receptor thereby limiting cyst growth and delaying the decline in kidney function. These treatment options also require repeated dosing and may cause critical side effects like hepatotoxicity and polyuria. Cell and gene therapies (CGT) hold immense potential for treating ADPKD by directly targeting and correcting the underlying genetic cause responsible for cyst formation.

[0019] However, CGTs have their own challenges owing to the complexities involved in achieving delivering therapeutic cargo to target cell types in the kidney i.e. the renal epithelial cells, collecting ducts. The difficulty includes overcoming biological barriers like the glomerular filtration barrier and navigating the complex structure that has over 26 unique cell -types. Other challenges with respect to cell and gene therapies also include minimizing off-target effects and immune responses, thereby ensuring durable and safe gene expression, and addressing vector limitations such as cargo capacity and potential insertional mutagenesis. The efficient delivery of genetic payloads continues to be a major challenge in the advancement of therapeutic modalities. Viral vectors, though highly efficient, are constrained by issues such as immunogenicity and complex, resource-intensive manufacturing processes. As a result, lipid nanoparticles (LNPs) have emerged as promising non-viral alternative, offering advantages including reduced immune response, potential for re-dosing, and scalability in production.

[0020] However, the existing LNP platforms primarily achieve hepatic delivery, with limited success in targeting extra-hepatic tissues such as muscle. To expand the therapeutic reach of LNPs and enable broader clinical applications, key challenges must be addressed: improving extrahepatic delivery efficiency, enhancing biodegradability while minimizing toxicity at therapeutic doses, and developing robust, scalable synthesis methodologies.

[0021] Therefore, there is need to have lipid nanoparticles and formulations based on the same which are stable and achieve extra hepatic targeting but with significantly reduced cytotoxicity, better encapsulation efficiency thereby achieving higher transfection.

[0022] SUMMARY OF THE INVENTION

[0023] Present invention aims to provide a state-of-the-art lipid nanoparticle (LNP) and an LNP delivery platform designed to address the key challenges associated with the therapeutic payload delivery. This platform enables precise and efficient targeting of diverse tissues and cell types, facilitating safe and effective delivery of therapeutic payloads to previously inaccessible or minimally accessible organs.

[0024] Accordingly, the present invention relates to a lipid of Formula I or its pharmaceutically acceptable salt, tautomer, prodrug or stereoisomer thereof with the following structure: Ri

[0025]

[0026] R4

[0027] wherein Rs is

[0028] hydrogen; C1-C3 alkyl; C3-C7 cycloalkyl; aryl, heteroaryl or heterocycloalkyl; -(CH2)OC(R)2(CH2)0-18-OQ, -C(O)NQR or -(CH2)0-18Q, in which Q is H, OH, -NHC(S)N(R)2, -NHC(O)N(R)2, -N(R)C(O)R', -N(R)S(O)2R', -N(R)R', -NHC(=NR)N(R′), -NHC(=CHR)N(R′)2, -OC(O)N(R)2, -N(R)C(O)OR'; wherein R and R' are same or different and are selected from hydrogen; C1-C3 alkyl; C3-C7 cycloalkyl; aryl, heteroaryl or heterocycloalkyl,

[0029] or a group selected from:

[0030]

[0031] ^NH

[0032] O

[0033]

[0034] OH

[0035] OH

[0036] OH

[0037]

[0038] Wherein the ‘*’ denotes the point of attachment to the ‘G’ group.

[0039] G is a single, double or triple bond; unsubstituted or substituted C1-C3alkyl, -(CH2)0-18-; -O(C=O)-; -O(C=O)O; -O(C=S)O; S-S; -O-; -(C=O)S(CH2)0-18; -NH(C=O)-;

[0040] Z is either C orN;

[0041] X1, X2, X3and X4are selected from C, NH, O, or S provided at least two of X1, X2, X3and X4are O; Ai and A2 are each independently C 1-C3 alkylene group; Ai and A2 can optionally together with the Z-atom to which they are attached form a 5 membered ring;

[0042] Li and L2 are the same or different and are each independently selected from

[0043] substituted or unsubstituted C1-C12 alkylene or C1-C12 alkenylene;

[0044] -C(O)O-, -OC(O)-, -C(O)N(R")-, -P(O)(OR")O-;

[0045] (CH2)0-18-; -OC(O)(CH2)0-18-; -OC(0)0(CH2)O-I8-; -CH(OR")2-; -OCOCH(R")2-;

[0046] -C(O)O(CH2)0-18-;

[0047] -C(O)O(CH2)0-18(C2H2)0-18CH-;

[0048] -C(O)OCH-;

[0049] where R" is hydrogen and un / substituted C1-3 alkyl; -S-S-, an aryl group, and a heteroaryl group;

[0050] Ri, R2 are independently selected from the group consisting of hydrogen, straight chain or branched

[0051] Ci-Cis alkyl, Ci-Cis alkenyl, Ci-Cis alkynyl;

[0052] C0-C18 alkyl- D - Ci-Cis alkyl;

[0053] C0-C14 alkylene- D - Ci-Cis alkylene;

[0054] C0-C18 alkyl- D - Ci-Cis alkylene;

[0055] C0-C14 alkylene- D - Ci-Cis alkyl;

[0056] where D is -C(O)-; -COO-; -C(O)S-; -O-; -S-;

[0057] and further

[0058] Ri and R2 optionally together with the carbon atom to which they are attached form a 3-5 membered heterocyclic ring with RI and R2 being selected from O or S; wherein the 3-5 membered heterocycle ring is optionally substituted with straight chain or branched Ci-Cis alkyl, C1-C14 alkenyl, C0-C14 alkyl- D - Ci-Cis alkyl; C0-C14 alkylene- D - Ci-Cis alkylene;, C0-C14 alkyl- D - Ci-Cis alkylene;, C0-C14 alkylene- D - Ci-Cis alkyl; where D is -C(O)-; -COO-; -C(O)S-; -O-; -S-;

[0059] R3 and R4 are independently selected from the group consisting of H, straight chain or branched Ci-Cis alkyl, C1-C14 alkenyl, C0-C14 alkyl- D - Ci-Cis alkyl; C0-C14 alkylene- D -Ci-Cis alkylene;, C0-C14 alkyl-D- Ci-Cis alkylene;, C0-C14 alkylene-D- Ci-Cis alkyl; where D is -C(O)-; -COO-; -C(O)S-; -O-; -S-; and

[0060] m is 0-9; p is 0-9; b is 0-12; c is 0-12; n is 0-9, and n’ is 0-9.

[0061] In an embodiment, the present invention provides a compound of Formula 1-A,

[0062]

[0063] Formula 1-A

[0064] wherein Ai and A2 are each independently C1-C3 alkylene group; R 6 and R7 are independently selected from straight chain or branched Ci-Cis alkyl, C1-C14 alkenyl, C0-C14 alkyl- D - Ci-Cis alkyl; C0-C14 alkylene- D - Ci-Cis alkylene;, C0-C14 alkyl- D - Ci-Cis alkylene;, C0-C14 alkylene- D - Ci-Cis alkyl; where D is -C(O)-; -COO-; -C(O)S-; -O-; -S-;

[0065] and wherein R3, R4, Rs, m, p, b and c are as defined for formula (I).

[0066] In another embodiment, the present invention provides a compound of Formula 1-B,

[0067]

[0068] Formula 1-B

[0069] wherein

[0070] Ai and A2 are each independently C1-C3 alkylene group; Re and R7 are independently selected from straight chain or branched Ci-Cis alkyl, C1-C14 alkenyl, C0-C14 alkyl- D - Ci-Cis alkyl; C0-C14 alkylene- D - Ci-Cis alkylene;, C0-C14 alkyl- D - Ci-Cis alkylene;, C0-C14 alkylene- D - Ci-Cis alkyl; where D is -C(O)-; -COO-; -C(O)S-; -O-; -S-;

[0071] and wherein R3, R4, R5, m, p, b and c are as defined for formula (I).

[0072] In another embodiment, the present invention provides a compound of Formula 1-C,

[0073]

[0074] Formula 1-C

[0075] wherein

[0076] Ai and A2 are each independently C1-C3 alkylene group; Re and R7 are independently selected from straight chain or branched Ci-Cis alkyl, C1-C14 alkenyl, C0-C14 alkyl- D - Ci-Cis alkyl; C0-C14 alkylene- D - Ci-Cis alkylene;, C0-C14 alkyl- D - Ci-Cis alkylene;, C0-C14 alkylene- D - Ci-Cis alkyl; where D is -C(O)-; -COO-; -C(O)S-; -O-; -S-;

[0077] and wherein R3, R4, R5, m, p, b and c are as defined in formula (I).

[0078] In another embodiment, the present invention provides a compound of Formula 1-D,

[0079]

[0080] Formula 1-D

[0081] wherein Ai and A2 are each independently C1-C3 alkylene group; Re and R7 are independently selected from straight chain or branched Ci-Cis alkyl, C1-C14 alkenyl, C0-C14 alkyl- D - Ci-Cis alkyl; C0-C14 alkylene- D - Ci-Cis alkylene;, C0-C14 alkyl- D - Ci-Cis alkylene;, C0-C14 alkylene- D - Ci-Cis alkyl; where D is -C(O)-; -COO-; -C(O)S-; -O-; -S-;

[0082] and wherein R3, R4, R5, m, p, b and c are as defined in formula (I).

[0083] In another embodiment, the present invention provides a compound of Formula 1-E,

[0084]

[0085] Formula 1-E

[0086] wherein Ai and A2 are each independently C1-C3 alkylene group;

[0087] and wherein R3, R4, Rs, m, p, b and c are as defined in formula (I).

[0088] In another embodiment, the present invention provides a compound of Formula 1-F,

[0089]

[0090] R4

[0091] Formula 1-F

[0092] wherein AI and A2 are each independently C1-C3 alkylene group;

[0093] and wherein R3, R4, Rs, m, p, b and c are as defined in formula (I).

[0094] In another embodiment, the present invention provides a compound of Formula 1-G,

[0095]

[0096] Formula 1-G

[0097] wherein Ri and R2 are each independently hydrogen, Ci-Cis alkyl, Ci-Cis alkenyl, or Ci-Cis alkynyl;

[0098] and wherein R3, R4, R5, m, p, b and c are as defined in formula (I).

[0099] In another embodiment, the present invention provides a compound of Formula 1-H,

[0100]

[0101] Formula 1-H

[0102] wherein Ai and A2 are each independently C1-C3 alkylene group; Re and R7 are independently selected from straight chain or branched Ci-Cis alkyl, C1-C14 alkenyl, C0-C14 alkyl- D - Ci-Cis alkyl; C0-C14 alkylene- D - Ci-Cis alkylene;, C0-C14 alkyl- D - Ci-Cis alkylene;, C0-C14 alkylene- D - Ci-Cis alkyl; where D is -C(O)-; -COO-; -C(O)S-; -O-; -S-;

[0103] and wherein R3, R4, R5, m and p are as defined in formula (I).

[0104] In another embodiment, the present invention provides a compound of Formula 1-1,

[0105]

[0106] Formula 1-1

[0107] wherein Ai and A2 are each independently C1-C3 alkylene group; Re and R7 are independently selected from straight chain or branched Ci-Cis alkyl, C1-C14 alkenyl, C0-C14 alkyl- D - Ci-Cis alkyl; C0-C14 alkylene- D - Ci-Cis alkylene;, C0-C14 alkyl- D - Ci-Cis alkylene;, C0-C14 alkylene- D - Ci-Cis alkyl; where D is -C(O)-; -COO-; -C(O)S-; -O-; -S-;

[0108] and wherein R3, R4, Rs, m and p are as defined in formula (I).

[0109] In another embodiment, the present invention provides a compound of Formula 1-J,

[0110]

[0111] Formula 1-J

[0112] wherein Ai and A2 are each independently C1-C3 alkylene group; Re and R7 are independently selected from straight chain or branched Ci-Cis alkyl, C1-C14 alkenyl, C0-C14 alkyl- D - Ci-Cis alkyl; C0-C14 alkylene- D - Ci-Cis alkylene;, C0-C14 alkyl- D - Ci-Cis alkylene;, C0-C14 alkylene- D - Ci-Cis alkyl; where D is -C(O)-; -COO-; -C(O)S-; -O-; -S-;

[0113] and wherein R3, R4, R5, m and p are as defined in formula (I).

[0114] In another embodiment, the present invention provides a compound of Formula 1-K,

[0115] s.

[0116]

[0117] Formula 1-K wherein Ai and A2 are each independently C1-C3 alkylene group; Ri and R2 are each independently hydrogen, Ci-Cis alkyl, Ci-Cis alkenyl, or Ci-Cis alkynyl; or Ri and R2 optionally together with the carbon atom to which they are attached form a 5 membered heterocyclic ring with RI and R2 being selected from O; wherein the 5 membered heterocycle ring is optionally substituted with straight chain or branched Ci-Cis alkyl, C1-C14 alkenyl, Co-C14 alkyl- D - Ci-Cis alkyl; C0-C14 alkylene- D - Ci-Cis alkylene;, C0-C14 alkyl- D - Ci-Cis alkylene;, C0-C14 alkylene- D - Ci-Cis alkyl; where D is -C(O)-; -COO-; -C(O)S-; -O-; -S-; and wherein R3, R4, R5, m, b and c are as defined in formula (I).

[0118] In another embodiment, the present invention provides a compound of Formula 1-L,

[0119] Formula 1-L

[0120]

[0121] wherein Ai and A2 are each independently C1-C3 alkylene group; Ri and R2 are each independently hydrogen, Ci-Cis alkyl, Ci-Cis alkenyl, or Ci-Cis alkynyl; or Ri and R2 optionally together with the carbon atom to which they are attached form a 5 membered heterocyclic ring with RI and R2 being selected from O; wherein the 5 membered heterocycle ring is optionally substituted with straight chain or branched Ci-Cis alkyl, C1-C14 alkenyl, Co-C14 alkyl- D - Ci-Cis alkyl; C0-C14 alkylene- D - Ci-Cis alkylene;, C0-C14 alkyl- D - Ci-Cis alkylene;, C0-C14 alkylene- D - Ci-Cis alkyl; where D is -C(O)-; -COO-; -C(O)S-; -O-; -S-; and wherein R3, R4, R5, m, b and c are as defined in formula (I).

[0122] In another embodiment, the present invention provides a compound of Formula 1-M,

[0123] H

[0124] N

[0125]

[0126] 4Formula 1-M

[0127] wherein Ai and A2 are each independently C1-C3 alkylene group;

[0128] and wherein R3, R4, R5, m, p, b, and c are as defined in formula (I).

[0129] In a further aspect, the present invention relates to a process for the preparing the lipid of formula I.

[0130] In an aspect, the present invention also relates to a formulation comprising at least an ionizable lipid of formula (I), a cholesterol, one or more of PEGylated lipid or / and helper lipid. In some embodiments, the formulation comprises cationic lipid. In some embodiments, the formulation the formulation encapsulates a therapeutic agent for target cell delivery with selective kidney, heart, spleen, lung tropism, lymphatic tissue. In an embodiment the formulation can comprise 30 to 60% by weight of at least one ionizable lipid of Formula I, 35 to 50% by weight of cholesterol, 1 to 5% by weight of PEGylated lipid, 7 to 10% by weight of helper lipid.

[0131] In a further embodiment, the lipid formulation may comprise cationic lipid.

[0132] In another embodiment the formulation of the present invention comprises 30-50% by weight of at least one ionizable lipid of Formula I, 1-10 % by weight of cationic lipid, 35-45 % by weight of cholesterol, 1-10% by weight of PEGylated lipid and 5-15% by weight of helper lipid.

[0133] In another embodiment, the formulation can comprise 30 to 70% by weight of at least one ionizable lipid, 10 to 30% by weight of cholesterol, 15 to 30% by weight of one or more PEGylated and / or helper lipids.

[0134] In a specific embodiment the lipid formulation comprises a suitable ratio of all the components. Preferably the components, ionizable lipid: cationic lipid: DSPC: Choi: PEG are present in the ratio 41.67: 4.63: 9.4: 42.7: 1.6.

[0135] In a specific embodiment the lipid formulation comprises at least one lipid of Formula I, wherein the formulation encapsulates a therapeutic agent for target cell delivery beyond liver with delivery to other organs such as but not limited to kidney, heart, spleen, lung, lymphatic tissue.

[0136] In a further embodiment, the lipid formulation comprises at least one lipid of Formula I and encapsulates one or more therapeutic agents for target cell delivery to a diseased organ including but not limited the kidney, liver, lung, pancreas, or any other organs and target cell types including but not limited to epithelial, endothelial, stromal, and other specialized cell types, including kidney-associated proximal convoluted tubule, distal convoluted tubule, and collecting duct cells.

[0137] In a specific embodiment the lipid formulation comprises at least one lipid of Formula I and encapsulates a therapeutic agent for target cell delivery with selective cystic or fibrotic kidney tropism.

[0138] In yet another aspect, the present invention relates to a method of delivering the therapeutic agent comprising encapsulating a therapeutic agent in the lipid formulation comprising at least one lipid of Formula I and administering the encapsulated therapeutic agent into the body of a subject.

[0139] In an embodiment, the lipid formulation is administered through intravenous, intra-arterial, intraperitoneal, intraparenchymal, subcutaneous, intramuscular, intradermal, intrathecal, intraocular, inhalational, intranasal, buccal, sublingual, oral, transdermal, intrapulmonary, intralymphatic, intranodal, intratumoral, intracavitary, intratracheal, or catheter-based, deviceassisted, or image-guided administration.

[0140] In a specific embodiment wherein the lipid formulation is administered through intravenous (IV) route, renal artery injection, retrograde ureteral injection (RU), renal vein injection, peritoneal injection, subcapsular injection, intra-parenchymal injection to reach different cell types within the kidney.

[0141] In another embodiment, the therapeutic agent is a nucleic acid-based material such as RNA (including mRNA, saRNA, siRNA, shRNA, miRNA, and guide RNAs), DNA, anti-sense oligonucleotides, plasmids, gene-editing components, and nucleic acid analogs; proteins and peptides including enzymes, antibodies, antibody fragments, cytokines, growth factors, and peptide therapeutics; small molecules of natural, synthetic, or semi -synthetic origin; immunomodulatory agents including vaccines, adjuvants, immune stimulants, or immune suppressants; biologies such as viral vectors, fusion proteins, aptamers, and polysaccharides; diagnostic or imaging agents including contrast agents and reporter constructs; and prodrugs or precursor molecules capable of conversion to an active form in vivo.

[0142] In yet another aspect of the invention, there is provided a method of delivering a payload comprising encapsulating a therapeutic agent in the lipid formulation of the invention and administering the lipid formulation comprising the therapeutic agent into the body of a subject.

[0143] DRAWINGS

[0144] Figure 1: Comparison of encapsulation efficiencies of FC2 LNPs against F7, F14, F15 and F16 and FC2 LNPs Figure 2: 90-Day Stability data of LNPs containing acetal oxolane family ionizable lipids stored at 4oC, prepared via microfluidic mixing method. Top: Particle size and PDI; Bottom: Encapsulation Efficiency and Final cargo concentration

[0145] Figure 3a:: in vitro release of mCherry-encapsulated F7, F14, F15, F16, and FC2 LNPS at 2 pg / well concentration in WT9-7 cells, observed at 6 h, 24 h and 48 h

[0146] Figure 3b:: Cytotoxicity of mCherry encapsulated F7, F14, F15, F16, and FC2 LNPS at 2 pg / well concentration in WT9-7 cells observed at 6 h, 24 h and 48 h

[0147] Figure 4: Release (top) and Cytotoxicity (bottom) of mCherry encapsulated F1, F2, F3 LNPS at 1 pg / well concentration in WT9-7 cells observed at 24 h and 48 h

[0148] Figure 5: in vivo biodistribution of F7 LNP encapsulating FLuc mRNA at 0.5 mg / kg dose, 6 hrs after intravenous injection in BALB / c mice. Top Left: in vivo imaging Top Right: ex vivo imaging; Bottom: Quantification of luciferase expression in mice 6 hrs post intravenous injection

[0149] Figure 6: in vivo biodistribution of FC1, F4, F5, and F6 LNPs encapsulating FLuc mRNA at 0.5 mg / kg dose, 6 hrs after intravenous injection in BALB / c mice. 6a: in vivo imaging; 6b: ex vivo imaging; 6c: Quantification of luciferase expression

[0150] Figure 7: Tissue toxicity study

[0151] Figure 8: Left: In vivo and ex vivo biodistribution of F7 (0.4 mg / kg) administered via retrograde ureteral route, assessed using IVIS imaging at 6 hours post-injection in healthy BALB / c mice. Right: Organ-wise biodistribution for F7, radiance values calculated using IVIS images post 6 hrs.

[0152] Figure 9: in vivo biodistribution of F7 (0.4 mg / kg) 6 hrs post renal artery injection in healthy BALB / c mice. Left: in-vivo imaging. Right: ex vivo imaging

[0153] Figure 10: Top: In vivo and ex vivo biodistribution of F7 (0.4 mg / kg) administered via retrograde ureteral route in PKD1 model at 12 weeks of age, assessed using IVIS imaging at 6 hours post-injection. Bottom: Organ-wise biodistribution for F7, radiance values calculated using IVIS images post 6 hrs

[0154] Figure 11: Immunofluorescence staining of right kidney sections from PKD1 Pkdlfl / fl; Cdhl6-CreERT2ki / wt model mice at 13-14 weeks age, injected with F8 LNPs, showing colocalization of reporter protein fLuc (red) with collecting duct marker AQP2 and proximal tubular marker LRP2 (green). Note: the first row Bar=200 p

[0155] Figure 12: IHC of kidney, liver and spleen cross-sections of C57BL / 6 mice (6-8 weeks, male) 7 days post retrograde ureteral injection of F7 LNP compared to non-injected control

[0156] Figure 13: DIC images of kidney sections after renal artery injection in BALB / c mice, showing lower toxicity with F7 compared to FC2 LNP. Uninjected control included for reference

[0157] Figure 14: Quantification of relative gene editing efficiency of F13, FC3 and FC4 LNP mediated single gRNA transfection in TKPTS cells at 48 hr

[0158] DETAILED DESCRIPTION OF THE INVENTION

[0159] Definitions

[0160] For convenience, before further description of the present disclosure, certain terms employed in the specification, and examples are delineated here. These definitions should be read in the light of the remainder of the disclosure and understood as by a person of skill in the art. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0161] As used herein, the articles including "a" and "an" when used in a claim, are understood to mean one or more of what is claimed or described.

[0162] As used herein, the word “comprise”, and variations such as “comprises” and “comprising”, will be understood to imply the inclusion of a stated element or step or group of element or steps but not the exclusion of any other element or step or group of element or steps.

[0163] As used herein, the terms "include," "includes," and "including," are meant to be non-limiting and are understood to mean "comprise," "comprises," and "comprising," respectively. As used herein, the terms “one embodiment” or “an embodiment” means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, the appearances of the phrases “in one embodiment” or “in an embodiment” in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

[0164] The term “lipid” refers to a group of organic compounds that include, but are not limited to, esters of fatty acids and are generally characterized by being poorly soluble in water, but soluble in many organic solvents. Lipids are usually divided into at least three classes: (1) “simple lipids,” which include fats and oils as well as waxes; (2) “compound lipids,” which include phospholipids and glycolipids; and (3) “derived lipids” such as steroids. Lipids may be ionizable as well as or cationic in nature, or a salt or isomer thereof, preferably a pharmaceutically acceptable salt.

[0165] An “ionizable lipid” refers to an amphiphilic molecule comprising at least one ionizable head group and one or more hydrophobic tails, designed to reversibly acquire a net positive charge under acidic conditions while remaining neutral or minimally charged at physiological pH. The ionizable lipid facilitates the encapsulation of anionic biomolecules, such as nucleic acids, by forming stable complexes under acidic conditions and promotes efficient intracellular delivery by enabling endosomal escape through pH-triggered transitions. The lipid may include modifications to its head group, linker, or tail regions to optimize properties such as encapsulation efficiency, biodegradability, pharmacokinetics, and biodistribution.

[0166] A “cationic lipid” refers to structures bearing a positive charge, either permanently, or not permanently but in response to certain conditions such as pH. Thus, the term “cationic” covers both “permanently cationic” and “cationisable”. As used herein, “permanently cationic” means that the respective compound, or group or atom, is positively charged at any pH value or hydrogen ion activity of its environment. Typically, the positive charge is results from the presence of a quaternary nitrogen atom. Where a compound carries a plurality of such positive charges, it may be referred to as permanently polycationic, which is a subcategory of permanently cationic. A “nucleic acid payload” refers to any natural, synthetic, or chemically modified nucleic acid molecule, or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer, tautomer, derivative, conjugate, or complex thereof, that is encapsulated, associated, or delivered using a lipid nanoparticle (LNP) or other delivery system. Exemplary nucleic acid payloads include, but are not limited to: messenger RNA (mRNA), self-amplifying RNA (saRNA), circular RNA (circRNA), small interfering RNA (siRNA), microRNA (miRNA), antisense oligonucleotides (ASOs), guide RNAs (gRNAs) for genome editing, CRISPR-associated ribonucleoprotein complexes (CRISPR RNPs), plasmid DNA (pDNA), DNA oligonucleotides, ribozymes, aptamers, and combinations thereof. The nucleic acid payload may be unmodified or chemically modified, such as by incorporation of modified nucleosides, phosphorothioate linkages, cap analogs, base modifications, or conjugation with targeting ligands, to enhance stability, translation efficiency, nuclease resistance, cellular uptake, or tissue specificity, thereby optimizing therapeutic performance.

[0167] A “pharmaceutically acceptable salt” refers to any salt form of an ionizable or cationic lipid that is non-toxic, physiologically compatible, and suitable for use in a pharmaceutical composition, including lipid nanoparticles (LNPs) for nucleic acid delivery. Such salts retain the lipid’s functional ability to encapsulate, protect, and deliver nucleic acid payloads, while modulating solubility, stability, or formulation properties. Exemplary pharmaceutically acceptable salts of ionizable or cationic lipids include acid addition salts formed with inorganic acids (e.g., hydrochloric, hydrobromic, sulfuric, phosphoric, nitric acids) or organic acids (e.g., acetic, citric, fumaric, maleic, tartaric, methanesulfonic, p-toluenesulfonic, or benzenesulfonic acids), as well as base addition salts formed with inorganic bases (e.g., sodium, potassium, calcium, magnesium, or ammonium hydroxides) or organic bases (e.g., ethanolamine, diethanolamine, tromethamine, or lysine). This definition also encompasses solvates, hydrates, polymorphs, stereoisomers, tautomers, and other pharmaceutically acceptable derivatives of such salts.

[0168] As used herein, “helper lipid” refers to a lipid component that, when incorporated into a lipid nanoparticle (LNP) or other lipid-based delivery system, contributes to particle stability, structural integrity, self-assembly, or delivery efficiency without necessarily serving as the primary carrier of a therapeutic payload. Helper lipids include, without limitation, neutral, zwitterionic, or structural phospholipids such as DOPE, DOPC, DSPC, DSPE, phosphatidylserine, phosphatidylglycerol, cholesterol, and derivatives thereof, and encompass natural or synthetic lipids, as well as pharmaceutically acceptable salts, solvates, hydrates, stereoisomers, tautomers, analogs, and derivatives. The term further includes lipid conjugates formed by covalent attachment of peptides (including targeting or cell-penetrating peptides), oligonucleotides, small molecules, antibodies or antibody fragments, or other functional moieties via cleavable or non-cleavable linkers.

[0169] A “PEGylated lipid” refers to a lipid molecule that is covalently conjugated to a polyethylene glycol (PEG) moiety or a derivative thereof, or a pharmaceutically acceptable salt, solvate, hydrate, stereoisomer, tautomer, or derivative thereof. PEGylated lipids are typically included in lipid nanoparticles (LNPs) or other lipid-based delivery systems to modulate particle size, improve colloidal stability, reduce aggregation, and extend circulation time in vivo by providing a steric barrier that decreases nonspecific protein adsorption and recognition by the mononuclear phagocyte system. Exemplary PEGylated lipids include, without limitation, phospholipid-PEG conjugates such as DMPE-PEG and DSPE-PEG; stearic-acid-PEG derivatives; and mono-, di-, or multi-substituted PEG-lipid conjugates bearing alkyl, alkenyl, or alkynyl chains ranging from C6 to C22. The term further encompasses PEGylated cholesterol derivatives and other natural or synthetic lipid-PEG conjugates suitable for incorporation into lipid-based delivery systems. In certain embodiments, the PEG moiety has a molecular weight ranging from about 500 Da to about 5 kDa, including commonly used PEGs of approximately 1 kDa, 2 kDa, or 5 kDa, without limitation to higher or lower molecular weights as required by the formulation. In certain embodiments, a PEGylated lipid may be further chemically modified or conjugated to a biomolecule or functional moiety — including, without limitation, peptides (such as targeting, receptor-binding, or cell-penetrating peptides), oligonucleotides (including DNA, RNA, siRNA, or antisense constructs), small molecules, or antibodies or antibody fragments — through cleavable or non-cleavable linkers, esterification, amidation, click chemistry, or other coupling strategies known in the art. Such conjugated PEGylated lipids remain within the scope of this definition provided they retain the ability to modulate steric stabilization, nanoparticle assembly, colloidal stability, or delivery performance of the lipid-based formulation.

[0170] The term “cholesterol” refers to a sterol or sterol derivative that can be incorporated into lipid nanoparticles (LNPs) or other lipid-based delivery systems to modulate membrane fluidity, stability, and structural integrity of the particle. Cholesterol and its derivatives can influence lipid packing, particle rigidity, encapsulation efficiency, biodistribution, and intracellular delivery of nucleic acid payloads. Exemplary cholesterol derivatives include, but are not limited to, cholesteryl esters, cholesteryl ethers, cholesteryl hemisuccinate, and other modified or functionalized cholesterol molecules. The term also encompasses natural or synthetic sterols, or pharmaceutically acceptable salts, solvates, hydrates, stereoisomers, tautomers, and derivatives thereof.

[0171] The term “therapeutic agent” refers to any compound, molecule, entity, or composition that produces, facilitates, or is intended to produce a beneficial biological, physiological, or pharmacological effect in a subject. The term encompasses agents for the treatment, mitigation, prevention, diagnosis, modulation, or monitoring of a disease, disorder, or biological condition. Therapeutic agents include, without limitation, nucleic acid-based materials such as RNA (including mRNA, saRNA, siRNA, shRNA, miRNA, and guide RNAs), DNA, plasmids, geneediting components, and nucleic acid analogs; proteins and peptides including enzymes, antibodies, antibody fragments, cytokines, growth factors, and peptide therapeutics; small molecules of natural, synthetic, or semi-synthetic origin; immunomodulatory agents including vaccines, adjuvants, immune stimulants, or immune suppressants; biologies such as viral vectors, fusion proteins, aptamers, and polysaccharides; diagnostic or imaging agents including contrast agents and reporter constructs; and prodrugs or precursor molecules capable of conversion to an active form in vivo. The term is intended to be interpreted broadly and includes any active moiety that may be encapsulated, associated, conjugated, or otherwise incorporated into the lipid nanoparticle formulations described herein.

[0172] In certain embodiments, the LNPs may further include active components incorporated, conjugated, or otherwise associated with the nanoparticle, such as small molecules, peptides, conjugated peptides, or antibodies, in an amount ranging from approximately 1-20% by weight. The invention also contemplates pre-formulated active moieties intended for incorporation into the LNPs during post-formulation processing.

[0173] The acronym “Autosomal Dominant Polycystic Kidney Disease (ADPKD)” refers to a hereditary systemic disorder characterized by the progressive development of numerous epithelial-lined cysts in both kidneys, resulting from mutations in the PKD1 or PKD2 genes. These mutations disrupt the function of polycystin proteins, leading to abnormal tubular cell proliferation, fluid secretion, and extracellular matrix remodeling. The term “tropism” in the context of drug or therapeutic delivery, refers to the selective targeting or preferential accumulation of a drug, biologic, or therapeutic agent within a specific tissue, organ, or cell type.

[0174] The present disclosure relates to novel lipids of Formula I including stereoisomers, pharmaceutically acceptable salts or tautomers thereof, which can be used alone or in combination with other lipid components such as cationic lipids, sterols, PEGylated lipids, helper lipids and / or their analogs, and / or polymer conjugated lipids to form lipid nanoparticles for the delivery of therapeutic agents.

[0175] The present invention discloses lipids focussed on extra-hepatic taigeting, combining specialized linkers and pH-responsive ionization shifts to reduce liver uptake and enhance delivery to non-liver tissues.

[0176] The present invention discloses lipids with an optimized pKa for targeting the kidney and include a head group that coordinates with the payload, stabilizing it in circulation and reducing off-target interactions beyond the liver. Additionally, a closed acetal linker in the ionizable lipid further stabilizes the complex, resisting premature degradation and maintaining the LNP’s structure. These features, along with short, branched lipid tails that support encapsulation, enable controlled release and efficient endocytosis by kidney cells.

[0177] The novel lipids disclosed in the present invention also undeigo a pH-sensitive shift from neutral to anionic, which favour non-liver interactions due to their anionic profile under specific pH conditions. This shift lowers liver cell affinity, as liver tissues generally absorb neutral or positively charged particles, and promotes kidney uptake, where neutral and anionic particles are efficiently filtered. After endocytosis and payload release, the novel lipids of the present invention degrade into neutral byproducts, reducing toxicity compared to commercial lipids, which often degrade into cytotoxic ionic byproducts. Together, these features create a targeted delivery system that ensures extended circulation, precise extra-hepatic delivery, and reduced toxicity.

[0178] In an embodiment, the present disclosure provides a method for delivering / administering a therapeutic agent to a patient in need thereof, the method comprising preparing a composition of lipids comprising the lipids of Formula I and a therapeutic agent and delivering the composition to the patient.

[0179] In an aspect of the invention, the present disclosure pertains to novel lipids of Formula I include stereoisomers, pharmaceutically acceptable salts or tautomers thereof that are pH sensitive cationic lipids and unexpectedly achieve selective organ tropism.

[0180] Accordingly, the present invention relates to a lipid of Formula I or its pharmaceutically acceptable salt, tautomer, prodrug or stereoisomer thereof with the following structure:

[0181] G'

[0182] R2

[0183] X.

[0184] / *4

[0185]

[0186] Formula I

[0187] wherein Rs is

[0188] hydrogen; C1-C3 alkyl; C3-C7 cycloalkyl; aryl, heteroaryl or heterocycloalkyl; - (CH2)OC(R)2(CH2)O-IS-OQ, -C(O)NQR or -(CH2)0-18Q, in which Q is H, OH, -NHC(S)N(R)2, -NHC(O)N(R)2, -N(R)C(O)R', -N(R)S(O)2R', -N(R)R', -NHC(=NR)N(R′), -NHC(=CHR)N(R′)2, -OC(O)N(R)2, -N(R)C(O)OR'; wherein R and R' are same or different and are selected from hydrogen; C1-C3 alkyl; C3-C7 cycloalkyl; aryl, heteroaryl or heterocycloalkyl,

[0189] or a group selected from: ^NH

[0190] O

[0191]

[0192]

[0193] OH

[0194] OH

[0195] OH

[0196]

[0197] Wherein the ‘*’ denotes the point of attachment to the ‘G’ group.

[0198] G is a single, double or triple bond; unsubstituted or substituted C1-C3alkyl, -(CH2)0-18-; -O(C=O)-; -O(C=O)O; -O(C=S)O; S-S; -O-; -(C=O)S(CH2)0-18; -NH(C=O)-;

[0199] Z is either C orN;

[0200] X1, X2, X3and X4are selected from C, NH, O, or S provided at least two of X1, X2, X3and X4are O; A i and A2 are each independently C 1 -C3 alkylene group; A 1 and A2 can optionally together with the Z-atom to which they are attached form a 5 membered ring;

[0201] Li and L2 are the same or different and are each independently selected from

[0202] substituted or unsubstituted C1-C12 alkylene or C1-C12 alkenylene;

[0203] -C(O)O-, -OC(O)-, -C(O)N(R")-, -P(O)(OR")O-;

[0204] (CH2)0-18-; -OC(O)(CH2)0-18-; -OC(0)0(CH2)O-I8-; -CH(OR")2-; -OCOCH(R")2-;

[0205] -C(O)O(CH2)0-18-;

[0206] -C(O)O(CH2)0-18(C2H2)0-18CH-;

[0207] -C(O)OCH-;

[0208] where R" is hydrogen and un / substituted C1-3 alkyl; -S-S-, an aryl group, and a heteroaryl group;

[0209] Ri, R2 are independently selected from the group consisting of hydrogen, straight chain or branched

[0210] Ci-Cis alkyl, Ci-Cis alkenyl, Ci-Cis alkynyl;

[0211] C0-C18 alkyl- D - Ci-Cis alkyl;

[0212] C0-C14 alkylene- D - Ci-Cis alkylene;

[0213] C0-C18 alkyl- D - Ci-Cis alkylene;

[0214] C0-C14 alkylene- D - Ci-Cis alkyl;

[0215] where D is -C(O)-; -COO-; -C(O)S-; -O-; -S-;

[0216] and further

[0217] Ri and R2 optionally together with the carbon atom to which they are attached form a 3-5 membered heterocyclic ring with RI and R2 being selected from O or S; wherein the 3-5 membered heterocycle ring is optionally substituted with straight chain or branched Ci-Cis alkyl, C1-C14 alkenyl, C0-C14 alkyl- D - Ci-Cis alkyl; C0-C14 alkylene- D - Ci-Cis alkylene;, C0-C14 alkyl- D - Ci-Cis alkylene;, C0-C14 alkylene- D - Ci-Cis alkyl; where D is -C(O)-; -COO-; -C(O)S-; -O-; -S-;

[0218] R3 and R4 are independently selected from the group consisting of H, straight chain or branched Ci-Cis alkyl, C1-C14 alkenyl, C0-C14 alkyl- D - Ci-Cis alkyl; C0-C14 alkylene- D -Ci-Cis alkylene;, C0-C14 alkyl-D- Ci-Cis alkylene;, C0-C14 alkylene-D- Ci-Cis alkyl; where D is -C(O)-; -COO-; -C(O)S-; -O-; -S-; and

[0219] m is 0-9; p is 0-9; b is 0-12; c is 0-12; n is 0-9, and n’ is 0-9. Some non-limiting exemplary embodiments of the novel lipids of Formula (I) of the present disclosure are described in further detail below:

[0220] In an embodiment, the present invention provides a compound of Formula 1-A,

[0221]

[0222] Formula 1-A

[0223] wherein Ai and A2 are each independently C1-C3 alkylene group; R 6 and R7 are independently selected from straight chain or branched Ci-Cis alkyl, C1-C14 alkenyl, C0-C14 alkyl- D - Ci-Cis alkyl; C0-C14 alkylene- D - Ci-Cis alkylene;, C0-C14 alkyl- D - Ci-Cis alkylene;, C0-C14 alkylene- D - Ci-Cis alkyl; where D is -C(O)-; -COO-; -C(O)S-; -O-; -S-;

[0224] and wherein R3, R4, Rs, m, p, b and c are as defined for formula (I).

[0225] In another embodiment, the present invention provides a compound of Formula 1-B,

[0226]

[0227] Formula 1-B

[0228] wherein

[0229] Ai and A2 are each independently C1-C3 alkylene group; Re and R7 are independently selected from straight chain or branched Ci-Cis alkyl, C1-C14 alkenyl, C0-C14 alkyl- D - Ci-Cis alkyl; C0-C14 alkylene- D - Ci-Cis alkylene;, C0-C14 alkyl- D - Ci-Cis alkylene;, C0-C14 alkylene- D - Ci-Cis alkyl; where D is -C(O)-; -COO-; -C(O)S-; -O-; -S-;

[0230] and wherein R3, R4, R5, m, p, b and c are as defined for formula (I). In another embodiment, the present invention provides a compound of Formula 1-C,

[0231]

[0232] Formula 1-C

[0233] wherein

[0234] Ai and A2 are each independently C1-C3 alkylene group; Re and R7 are independently selected from straight chain or branched Ci-Cis alkyl, C1-C14 alkenyl, C0-C14 alkyl- D - Ci-Cis alkyl; C0-C14 alkylene- D - Ci-Cis alkylene;, C0-C14 alkyl- D - Ci-Cis alkylene;, C0-C14 alkylene- D - Ci-Cis alkyl; where D is -C(O)-; -COO-; -C(O)S-; -O-; -S-;

[0235] and wherein R3, R4, R5, m, p, b and c are as defined in formula (I).

[0236] In another embodiment, the present invention provides a compound of Formula 1-D,

[0237]

[0238] Formula 1-D

[0239] wherein Ai and A2 are each independently C1-C3 alkylene group; Re and R7 are independently selected from straight chain or branched Ci-Cis alkyl, C1-C14 alkenyl, C0-C14 alkyl- D - Ci-Cis alkyl; C0-C14 alkylene- D - Ci-Cis alkylene;, C0-C14 alkyl- D - Ci-Cis alkylene;, C0-C14 alkylene- D - Ci-Cis alkyl; where D is -C(O)-; -COO-; -C(O)S-; -O-; -S-;

[0240] and wherein R3, R4, R5, m, p, b and c are as defined in formula (I). In another embodiment, the present invention provides a compound of Formula 1-E,

[0241]

[0242] Formula 1-E

[0243] wherein Ai and A2 are each independently C1-C3 alkylene group;

[0244] and wherein R3, R4, Rs, m, p, b and c are as defined in formula (I).

[0245] In another embodiment, the present invention provides a compound of Formula 1-F,

[0246]

[0247] R4

[0248] Formula 1-F

[0249] wherein Ai and A2 are each independently C1-C3 alkylene group; and wherein R3, R4, Rs, m, p, b and c are as defined in formula (I).

[0250] In another embodiment, the present invention provides a compound of Formula 1-G,

[0251]

[0252] Formula 1-G

[0253] wherein Ri and R2 are each independently hydrogen, Ci-Cis alkyl, Ci-Cis alkenyl, or Ci-Cis alkynyl;

[0254] and wherein R3, R4, R5, m, p, b and c are as defined in formula (I).

[0255] In another embodiment, the present invention provides a compound of Formula 1-H,

[0256]

[0257] Formula 1-H

[0258] wherein Ai and A2 are each independently C1-C3 alkylene group; Re and R7 are independently selected from straight chain or branched Ci-Cis alkyl, C1-C14 alkenyl, C0-C14 alkyl- D - Ci-Cis alkyl; C0-C14 alkylene- D - Ci-Cis alkylene;, C0-C14 alkyl- D - Ci-Cis alkylene;, C0-C14 alkylene- D - Ci-Cis alkyl; where D is -C(O)-; -COO-; -C(O)S-; -O-; -S-;

[0259] and wherein R3, R4, R5, m and p are as defined in formula (I).

[0260] In another embodiment, the present invention provides a compound of Formula 1-1,

[0261]

[0262] Formula 1-1

[0263] wherein Ai and A2 are each independently C1-C3 alkylene group; Re and R7 are independently selected from straight chain or branched Ci-Cis alkyl, C1-C14 alkenyl, C0-C14 alkyl- D - Ci-Cis alkyl; C0-C14 alkylene- D - Ci-Cis alkylene;, C0-C14 alkyl- D - Ci-Cis alkylene;, C0-C14 alkylene- D - Ci-Cis alkyl; where D is -C(O)-; -COO-; -C(O)S-; -O-; -S-;

[0264] and wherein R3, R4, Rs, m and p are as defined in formula (I).

[0265] In another embodiment, the present invention provides a compound of Formula 1-J,

[0266]

[0267] Formula 1-J

[0268] wherein Ai and A2 are each independently C1-C3 alkylene group; Re and R7 are independently selected from straight chain or branched Ci-Cis alkyl, C1-C14 alkenyl, C0-C14 alkyl- D - Ci-Cis alkyl; C0-C14 alkylene- D - Ci-Cis alkylene;, C0-C14 alkyl- D - Ci-Cis alkylene;, C0-C14 alkylene- D - Ci-Cis alkyl; where D is -C(O)-; -COO-; -C(O)S-; -O-; -S-;

[0269] and wherein R3, R4, R5, m and p are as defined in formula (I).

[0270] In another embodiment, the present invention provides a compound of Formula 1-K,

[0271] s.

[0272]

[0273] Formula 1-K

[0274] wherein Ai and A2 are each independently C1-C3 alkylene group; Ri and R2 are each independently hydrogen, Ci-Cis alkyl, Ci-Cis alkenyl, or Ci-Cis alkynyl; or Ri and R2 optionally together with the carbon atom to which they are attached form a 5 membered heterocyclic ring with RI and R2 being selected from O; wherein the 5 membered heterocycle ring is optionally substituted with straight chain or branched Ci-Cis alkyl, C1-C14 alkenyl, Co-C14 alkyl- D - Ci-Cis alkyl; C0-C14 alkylene- D - Ci-Cis alkylene;, C0-C14 alkyl- D - Ci-Cis alkylene;, C0-C14 alkylene- D - Ci-Cis alkyl; where D is -C(O)-; -COO-; -C(O)S-; -O-; -S-; and wherein R3, R4, Rs, m, b and c are as defined in formula (I).

[0275] In another embodiment, the present invention provides a compound of Formula 1-L,

[0276]

[0277] 0

[0278] Formula 1-L

[0279] wherein Ai and A2 are each independently C1-C3 alkylene group; Ri and R2 are each independently hydrogen, Ci-Cis alkyl, Ci-Cis alkenyl, or Ci-Cis alkynyl; or Ri and R2 optionally together with the carbon atom to which they are attached form a 5 membered heterocyclic ring with RI and R2 being selected from O; wherein the 5 membered heterocycle ring is optionally substituted with straight chain or branched Ci-Cis alkyl, C1-C14 alkenyl, Co-C14 alkyl- D - Ci-Cis alkyl; C0-C14 alkylene- D - Ci-Cis alkylene;, C0-C14 alkyl- D - Ci-Cis alkylene;, C0-C14 alkylene- D - Ci-Cis alkyl; where D is -C(O)-; -COO-; -C(O)S-; -O-; -S-; and wherein R3, R4, R5, m, b and c are as defined in formula (I).

[0280] In another embodiment, the present invention provides a compound of Formula 1-M,

[0281]

[0282] wherein Ai and A2 are each independently C1-C3 alkylene group;

[0283] and wherein R3, R4, Rs, m, p, b, and c are as defined in formula (I).

[0284] In a further aspect, the present invention relates to a process for the preparing the lipid of formula I. In another aspect, the present invention relates to a formulation comprising at least one lipid of Formula I, at least one PEGylated lipid, at least one helper lipid or cholesterol.

[0285] In another embodiment the lipid formulation comprises at least one ionizable lipid of formula (I), a cholesterol, one or more of PEGylated lipid or / and helper lipid.

[0286] In yet another embodiment, the formulation of the present invention comprises at least one lipid of Formula I, at least one PEGylated lipid and cholesterol. In one more embodiment, the formulation of the present invention comprises at least one lipid of Formula I, at least one PEGylated lipid and one helper lipid. As used herein, references to ‘the lipid formulation’ or ‘the composition’ are intended to encompass any of the foregoing embodiments, including formulations with or without a cationic lipid, helper lipid, PEGylated lipid, cholesterol, and / or one or more targeting moieties, unless otherwise specified.

[0287] In another embodiment, the formulation of the present invention comprises at least one ionizable lipid such as a lipid of Formula I, at least one PEGylated lipid, cholesterol and at least one helper lipid. In an embodiment the formulation can comprise 30 to 60% by weight of at least one ionizable lipid of Formula I, 35 to 50% by weight of cholesterol, 1 to 5% by weight of PEGylated lipid, 7 to 10% by weight of helper lipid.

[0288] In a further embodiment, the lipid formulation may comprise cationic lipid. In another embodiment the formulation of the present invention comprises 30-50% by weight of at least one ionizable lipid of Formula I, 1-10 % by weight of cationic lipid, 35-45 % by weight of cholesterol, 1-10% by weight of PEGylated lipid and 5-15% by weight of helper lipid. In another embodiment, the formulation can comprise 30 to 70% by weight of at least one ionizable lipid, 10 to 30% by weight of cholesterol, 15 to 30% by weight of one or more PEGylated and / or helper lipids.

[0289] In a specific embodiment the lipid formulation comprises a suitable ratio of all the components. Preferably the components, ionizable lipid: cationic lipid: DSPC: Choi: PEG are present in the ratio 41.67: 4.63: 9.4: 42.7: 1.6.

[0290] Any of the formulations described herein may be configured for delivery of an active agent to extrahepatic tissues, including but not limited to the spleen, kidney, lung, brain, bone marrow, lymph nodes, or peripheral tissues. In some embodiments, the compositions are capable of delivering the active agent to immune cell subsets such as dendritic cells, macrophages, monocytes, lymphocytes, or other antigen-presenting cells. Any of the formulations described herein may further include targeting moieties selected from small molecules, peptides, conjugated peptides, antibodies, antibody fragments, or other ligands configured to associate with the extracellular surface of the nanoparticle.

[0291] In another embodiment, the lipid formulation comprising at least one lipid of Formula I and encapsulating therapeutic agents / payloads for target cell delivery. In an embodiment the target cell delivery relates to delivery of the payload to the target cells, which include but are not limited to proximal convoluted tubules (PCT); distal convoluted tubules (DCT) or the collecting ducts of the kidney.

[0292] In one embodiment, the lipid formulation comprising at least one lipid of Formula I selectively delivers the therapeutic payload to an extra-hepatic organ, including but not limited to the kidney, spleen, pancreas, lung, heart, or lymphatic tissue. In certain embodiments, organ / tissue tropism is specifically accomplished by reducing or eliminating liver targeting (de-targeting the liver). In further embodiments, the lipid formulation selectively delivers the payload to specific cell types within said extra-hepatic organs, including but not limited to epithelial, endothelial, stromal, and other specialized cell types, including kidney-associated proximal convoluted tubule, distal convoluted tubule, and collecting duct cells. In a specific embodiment the lipid formulation comprises at least one lipid of Formula I, wherein the formulation encapsulates a therapeutic agent for target cell delivery beyond liver with delivery to other organs such as but not limited to kidney, heart, spleen, lung, lymphatic tissue. In a further embodiment, the lipid formulation comprises at least one lipid of Formula I and encapsulates one or more therapeutic agents for target cell delivery to a diseased organ including but not limited the kidney, liver, lung, pancreas, or any other organs and target cell types including but not limited to epithelial, endothelial, stromal, and other specialized cell types, including kidney-associated proximal convoluted tubule, distal convoluted tubule, and collecting duct cells. In a specific embodiment the lipid formulation comprises at least one lipid of Formula I and encapsulates a therapeutic agent for target cell delivery with selective cystic or fibrotic kidney tropism. In yet another aspect, the present invention relates to a formulation comprising of at least one lipid of Formula I delivering one or more payload(s) including but not limited to nucleic acidbased materials such as RNA (including mRNA, saRNA, siRNA, shRNA, miRNA, and guide RNAs), DNA, plasmids, gene-editing components, and nucleic acid analogs; proteins and peptides including enzymes, antibodies, antibody fragments, cytokines, growth factors, and peptide therapeutics; small molecules of natural, synthetic, or semi -synthetic origin; immunomodulatory agents including vaccines, adjuvants, immune stimulants, or immune suppressants; biologies such as viral vectors, fusion proteins, aptamers, and polysaccharides; diagnostic or imaging agents including contrast agents and reporter constructs; and prodrugs or precursor molecules capable of conversion to an active form in vivo.

[0293] In yet another aspect, the present invention relates to a method of delivering the therapeutic agent comprising encapsulating a therapeutic agent in the lipid formulation comprising at least one lipid of Formula I and administering the encapsulated therapeutic agent into the body of a subject.

[0294] The present invention possesses broad tunability for multiple organ systems, including kidney, which has a complex biology and has a substantial unmet medical need within this domain. The kidney’s highly specialized structure comprising distinct cell populations such as tubular epithelial cells, podocytes, and mesangial cells, presents considerable challenges for therapeutic intervention while simultaneously offering significant opportunities for targeted treatment. Through the development of LNPs engineered for precise delivery to specific renal cell types, the present invention demonstrates the potential to address a wide spectrum of kidney diseases.

[0295] The novel lipids of the present invention offer improved liver de-targeting and enhanced kidney biodistribution, with enhanced safety and superior encapsulation efficiency compared to commercially available lipids, which are often limited by toxicity and suboptimal payload release at therapeutic doses.

[0296] Present invention encompasses delivery to kidneys via intravenous (IV) injections, and localized delivery to enhance target cell uptake. To achieve cell-specific uptake, particularly in proximal convoluted tubules (PCTs), distal convoluted tubules (DCTs), and collecting ducts, which represent the primary target cells, the localized delivery approach via the retrograde ureteral (RU) route amongst various routes has been developed. This method enables the use of lower LNP doses, thereby reducing the overall lipid burden and minimizing immunogenicity. These localized routes of delivery are very relevant for potential single dose, or infrequently re-dosed therapeutics.

[0297] Thus, the present invention enables uniform delivery of the LNPs to the cells of interest i.e. proximal tubular cells and collecting ducts where cysts predominantly arise. Particularly, collecting duct-derived cysts are the most common and have an adverse impact on kidney function. Targeting these tissues is essential to address both cyst formation and the associated pathological signalling that drives the disease. The present invention enables delivery through retrograde ureteral (RU) injection and effectively achieves delivery to proximal tubular cells and collecting ducts, opening up doors to develop safe and efficient therapies to solve for ADPKD thus overcoming the limitations of current treatments that focus solely on symptomatic management.

[0298] In an embodiment, the method of delivering a payload(s) comprises encapsulating a therapeutic agent(s) in the lipid formulation comprising at least one lipid of Formula I and administering the encapsulated therapeutic agent(s) or payload(s) as 3-component / 4-component / 5-component formulation to targeted organ- or tissue-specific cell populations. These populations may include, without limitation, epithelial, endothelial, stromal, and other specialized cell types, including kidney-associated proximal convoluted tubule, distal convoluted tubule, and collecting duct cells.

[0299] In a further embodiment, the method of delivering a payload comprises encapsulating a therapeutic agent(s) in the lipid formulation comprising at least one lipid of Formula I and administering the encapsulated therapeutic agent(s) or payload(s) to a diseased organ. The diseased organ may include, without limitation, the kidney, liver, lung, pancreas, or any other organs affected by cystic, degenerative, inflammatory, metabolic, or structural pathologies.

[0300] In yet another embodiment, the therapeutic agent encapsulated by the lipid formulation comprising at least one lipid of Formula I is a nucleic acid payload, including messenger RNA (mRNA), self-amplifying RNA (saRNA), circular RNA (circRNA), small interfering RNA (siRNA), antisense oligonucleotides (ASOs), CRISPR-associated guide RNAs (gRNAs), or ribonucleoprotein complexes (RNPs). In a further embodiment, the method of delivering a payload comprises encapsulating a therapeutic agent(s) in the lipid formulation comprising at least one lipid of Formula I and administering the encapsulated therapeutic agent(s) or payload(s) to a diseased organ. The diseased organ may include, without limitation, the kidney, liver, lung, pancreas, or any other organs affected by cystic, degenerative, inflammatory, metabolic, or structural pathologies. In an embodiment, the lipid formulation is administered through intravenous, intra-arterial, intraperitoneal, intraparenchymal, subcutaneous, intramuscular, intradermal, intrathecal, intraocular, inhalational, intranasal, buccal, sublingual, oral, transdermal, intrapulmonary, intralymphatic, intranodal, intratumoral, intracavitary, intratracheal, or catheter-based, deviceassisted, or image-guided administration.

[0301] In an embodiment of the method of delivery of the present invention, the mode of administering the encapsulated therapeutic agent or the payload is through retrograde ureteral injection (RU).

[0302] In an embodiment of the method of delivery of the present invention, the mode of administering the encapsulated therapeutic agent or the payload is through infusion into the renal artery via a catheter or injection.

[0303] In an embodiment of the method of delivery of the present invention, the mode of administering the encapsulated therapeutic agent or the payload is through retrograde infusion into the renal vein.

[0304] In an embodiment of the method of delivery of the present invention, the mode of administering the encapsulated therapeutic agent or the payload is through subcapsular injection into the parenchyma of the kidney.

[0305] In an embodiment of the method of delivery of the present invention, the mode of administering the encapsulated therapeutic agent or the payload is through intra-peritoneal injection of the kidney.

[0306] In an embodiment of the method of delivery of the present invention, the mode of administering the encapsulated therapeutic agent or the payload is through intra-parenchymal injection of kidney. In a specific embodiment wherein the lipid formulation is administered through intravenous (IV) route, renal artery injection, retrograde ureteral injection (RU), renal vein injection, peritoneal injection, subcapsular injection, intra-parenchymal injection to reach different cell types within the kidney.

[0307] In another embodiment, the therapeutic agent is a nucleic acid-based material such as RNA (including mRNA, saRNA, siRNA, shRNA, miRNA, and guide RNAs), DNA, anti-sense oligonucleotides, plasmids, gene-editing components, and nucleic acid analogs; proteins and peptides including enzymes, antibodies, antibody fragments, cytokines, growth factors, and peptide therapeutics; small molecules of natural, synthetic, or semi -synthetic origin; immunomodulatory agents including vaccines, adjuvants, immune stimulants, or immune suppressants; biologies such as viral vectors, fusion proteins, aptamers, and polysaccharides; diagnostic or imaging agents including contrast agents and reporter constructs; and prodrugs or precursor molecules capable of conversion to an active form in vivo.

[0308] Further non-limiting representative embodiments of the lipids of Formula 1-A are provided herewith in TABLE – 1

[0309] ■

[0310] Formula 1-A

[0311] Compound

[0312] Structure

[0313] No.

[0314] H3C / — '

[0315] A001

[0316] H3C \

[0317]

[0318] A002

[0319] I

[0320] / —N

[0321] A003

[0322] HO— / \ \ \ z ^^ z ^^

[0323] 0

[0324] 0

[0325] 1 I 1 o

[0326] A004

[0327] A005

[0328] A006

[0329] A007

[0330]

[0331]

[0332]

[0333] Further non-limiting representative embodiments of the lipids of Formula 1-B are provided herewith in TABLE – 2 R5 IN.. p R4Formula 1-B

[0334] Compound I

[0335] Structure

[0336] No. ""■2.

[0337] 0

[0338] %

[0339] \A °

[0340] 4

[0341] B001 / \ o o o o

[0342] / \ o o o o

[0343] B002

[0344] B003

[0345]

[0346]

[0347]

[0348] Further non-limiting representative embodiments of the lipids of Formula 1-C are provided

[0349]

[0350]

[0351] Further non-limiting representative embodiments of the lipids of Formula 1-D are provided

[0352]

[0353] Formula 1-D

[0354] Compound

[0355] Structure

[0356] No.

[0357] D001

[0358] O O O O

[0359] )0 0^ X. y

[0360] < O)0 0^"

[0361] a

[0362] D002

[0363] I 0 z

[0364] 0 A 1

[0365] HOX

[0366] D003 N— '

[0367] ^0

[0368] / ) - \

[0369] 0K J

[0370]

[0371] Further non-limiting representative embodiments of the lipids of Formula 1-E are provided herewith in TABLE – 5

[0372]

[0373] Further non-limiting representative embodiments of the lipids of Formula 1-F are provided

[0374]

[0375] Further non-limiting representative embodiments of the lipids of Formula 1-G are provided

[0376]

[0377]

[0378] Further non-limiting representative embodiments of the lipids of Formula 1-H are provided herewith in TABLE – 8

[0379]

[0380] Further non-limiting representative embodiments of the lipids of Formula 1-1 are provided herewith in TABLE – 9

[0381]

[0382] Further non-limiting representative embodiments of the lipids of Formula 1-J are provided herewith in TABLE – 10 Formula 1-J

[0383]

[0384] Further non-limiting representative embodiments of the lipids of Formula 1-K are provided

[0385]

[0386]

[0387] Further non-limiting representative embodiments of the lipids of Formula 1-L are provided

[0388]

[0389]

[0390] Further non-limiting representative embodiments of the lipids of Formula 1-M are provided

[0391]

[0392]

[0393] Some exemplary lipids of the present invention include but not limited to the following:

[0394] Formula 1-B

[0395]

[0396] Formula 1-C

[0397] Formula 1-D

[0398]

[0399] Formula 1-E Formula 1-F

[0400]

[0401] It is understood that any embodiment of the lipids of Formula I, as set forth above, and any specific substituent and / or variable in the lipids of Formula I, as set forth above, may be independently combined with other embodiments and / or substituents and / or variables of lipids of Formula I to form more embodiments of the inventions not specifically herein. It is also understood that in the present description, combinations of substituents and / or variables of the depicted formulae are permissible only if such contributions result in stable lipids and formulations.

[0402] The present invention further discloses LNP formulations comprising at least one lipid as per Formula I, along with a helper lipid, a cationic lipid, cholesterol and at least one PEGylated lipid. The LNP formulations also exhibit extra hepatic delivery tissue / organ tropism specifically detargeting the liver and specifically targetting the kidney. The accumulation of drug products, and other therapeutics in the liver is prevented by the LNPs and their formulations as dislcosed in the present invention. This is an improvement over LNPs of prior art which have a tendency to localize in the liver. The formulations of the present invention have demonstrated localization in kidneys along with significantly less cytotoxicity, favourable safety profile and high encapsulation efficiency resulting in greater scope for therapeutic translation in taregting the kidney...

[0403] EXAMPLES

[0404] The foregoing examples of the related art and limitations related therewith are intended to be illustrative and not exclusive. Other limitations of the related art will become apparent to those of skill in the art upon a reading of the specification and a study of the figures.

[0405] EXAMPLE 1: STAGEWISE PROCESS OF PREPARING LIPIDS OF FORMULA 1-A Stage-1: Preparation of 8-(benzyloxy)octan-l-ol

[0406]

[0407]

[0408] A 250 mL two-neck round bottom flask was evacuated and backfilled with nitrogen. To this, 1,8 -octanediol 1 (10.0 g, 68.38 mmol, 1.0 equiv) was taken and 150 mL dry tetrahydrofuran (THF) was added. Then the reaction mixture was cooled down to 0 °C, and to the stirring solution, NaH (60% dispersion in mineral oil, 2.6 g, 68.38 mmol, 1.0 equiv) was added in portion under positive nitrogen pressure. The resulting mixture was stirred at room temperature for 30 min and again cooled down to 0 °C. After that, tetrabutylammonium iodide (TBAI, 6.83mmol, 2.5 g, 0.1 equiv) was added followed by benzyl bromide (41.03 mmol, 7.0 gm, 0.6 equiv) was added for 15 min and the resulting solution was stirred at room temperature for 18 hrs. After complete consumption of A, as indicated by TLC, the reaction mixture was quenched with water (60 mL) and the aqueous layer was extracted with ethyl acetate (3 x 60 mL). The combined organic layers were dried over anhydrous Na₂SO₄ and evaporated under reduced pressure. The crude residue was purified by silica gel column chromatography using 35% ethyl acetate in hexane as eluent to afford Int-2, (8-(benzyloxy)octan-l-ol) 6.0 g,37 %), respectively, as pale-yellow oil.

[0409] 'H NMR (400 MHz, CDC13) 5: ppm 7.34-7.26 (m, 5H); 4.50(s, 2H);.3.63 (t, 2H, J = 6 H); 3.46 (t, 2H, J = 6.4 H); 1.64-1.52 (m, 3H); 1.40-1.24 (m, 9 H).

[0410] Stage-2: Synthesis of 8-(benzyloxy) octanal

[0411]

[0412] A 250 mL two-neck round bottom flask was evacuated and backfilled with nitrogen, to this 70 ml of MDC was added once again nitrogen purged thoroughly. Then the pyridinium chlorochromate (PCC) (6.5 g, 30.48 mmol, 1.2 equiv.) was added to above solution and stirred for 5min. To the above reaction mixture, solution of 8-(benzyloxy) octan-l-ol (2) (6.0 g, 25.40 mmol, 1 equiv) in dichloromethane (10 mL) was added slowly for 15 to 20 min. The reaction was stirred at room temperature for 2 to 4 hrs, after completion of reaction, reaction mixture was filtered through hyflow to remove the black insoluble material, eluting with dichloromethane. After evaporation of the filtrate, the crude was purified by silica gel column chromatography using 5% ethyl acetate in hexane as eluent to obtained aldehyde (3) [8-(benzyloxy) octanal (5.0g, 84.7%) as a colorless oil.

[0413] 1H NMR (CDC13): 'H NMR (400 MHz, CDC13) 5: ppm 9.76-9.75 (t, 1H, J=1.6); 7.37-7.28 (m, 5H); 4.50-4.49 (s, 2H);.3.52--3.47 (m, 2H); 2.57-2.53 (m,2H); 1.98-1.92 (m, 2H).

[0414] Stage-3 (i): Synthesis of Tetradecane-7, 8-diol

[0415]

[0416]

[0417] A mixture of 7-tetradecene (purchased from TCI.) (7.9 g, 40.22 mmol), 7.1 ml of 37% H2O2 and 49 ml of formic acid was stirred at the temperature 40-50° C for 7 hrs. The reaction mixture was concentrated under a rotary evaporator in a hot water bath at ~30 torr to remove most of the water and formic acid. The above crude is washed with water (10 Vol) and extracted with ethyl acetate (15Vol) followed by concentration on a rotary evaporator. Then, residue dissolved in 10% KOH in Methanol (50 ml) and 100 ml toluene and heated to reflux for 6 hrs. The reaction mixture concentrated and residue dissolved in water and diluted with 500 mL ethyl acetate and transferred to a separatory funnel. The oiganic layer was separated, and the aqueous layer was extracted 3 times (3x200 mL) with ethyl acetate. The ethyl acetate extracts were combined and dried over anhydrous Na₂SO₄ Filtration, followed by concentration on a rotary evaporator at reduced pressure. The above crude was purified using silica gel column chromatography using 12% ethyl acetate in hexane as eluent to obtain desired diol-(5) [ tetradecane-7, 8-diol] as white powder (7.1 g, 77%).

[0418] 'H NMR (400 MHz, CDC13) 5: ppm 4.98-4.89 (m, 1H); 3.72-3.68(m, 1H); 1.68-1.26 (m, 20 H); 0.87-0.81(m, 6H).

[0419] Stage-3(ii): Synthesis of [2-(7-(benzyloxy) heptyl)-4-hexyl-5-pentyl-l,3-dioxolane]

[0420]

[0421] A solution of diol-5 (1 gm, 4.34 mmol, 1 equiv), comp-3 (1.2 gm, 5.12 mmol, 1.2 eqiv) in toluene (20 ml) was added PTS A (100 mg, 0.1 equiv ) and the reaction mixture heated under reflux 90 to 100° C for overnight. After completion of the reaction cool the reaction mixture and evaporated, the above crude was purified using silica gel column chromatography using 2% ethyl acetate in hexane as eluent to obtain Int-(6) [2-(7-(benzyloxy) heptyl)-4-hexyl-5-pentyl- 1,3 -dioxolane]) as colorless oil (1.67 g, 89 %).

[0422] ’HNMR (400 MHz, CDC13) 5: ppm 7.34-7.26 (m,5 H); 5.09-4.85 (m, 1H); 4.49 (s, 2H); 4.01-3.88 (m, 2H); 3.55-3.43(m, 2H); 1.62-1.29 (m, 33 H); 0.90-0.86(m, 6H).

[0423] Stage-4: Synthesis of 7-(4,5-dihexyl-l,3-dioxolan-2-yl) heptan-l-ol

[0424] Exact Mass: 356.33

[0425]

[0426] A solution of [2-(7-(benzyloxy) heptyl)-4-hexyl-5-pentyl-l,3-dioxolane] Int-6 (5.0 gm, 11.21 mmol, 1.0 equiv) in Ethyl acetate (80 mL) was made and Pd / C (500 mg, 0.1 equiv) was added. The reaction mixture was purged with H2 gas and stirred under an atmosphere of H2 (applied through balloon fdled with hydrogen gas) for 12 h. After complete consumption of starting material as indicated by TLC, the reaction mixture was filtered through a pad of celite, and washed with ethyl acetate (150 mL). The filtrates were then concentrated under reduced pressure and the crude Int-7, [7-(4,5-dihexyl-l,3-dioxolan-2-yl)heptan-l-ol] (3.91 gm, 98%) taken as directly further stage.

[0427] 1HNMR(400 MHz, CDC13) 5: ppm 5.11-4.86 (m, 1H); 4.02-3.89 (m, 2H); 3.65-3.49(m, 2H); 2.11-1.25 (m, 39 H); 0.90-0.86(m, 6H).

[0428] Stage-5: Synthesis of 7-(4,5-dihexyl-l,3-dioxolan-2-yl)heptanal [Common A]

[0429]

[0430] A 250 mL two-neck round bottom flask was evacuated and backfilled with nitrogen, and to this 10 ml of MDC was added and once again nitrogen purged thoroughly. Then the pyridinium chlorochromate (PCC) (600 mg, 2.69 mmol, 1.2 equiv) was added to above solution and stirred for 5min. To the above reaction mixture, solution of 7-(4, 5 -dihexyl- 1,3 -di oxolan-2-yl) heptan-l-ol] (7) (800 mg, 2.24 mmol, 1 equiv) in dichloromethane (3 mL) was added slowly for 15 to 20 min. The reaction was stirred at room temp, for 2 to 18 hrs, after completion of reaction, reaction mixture was filtered through hyflow to remove the black insoluble material, eluting with di chloromethane. After evaporation of the filtrate, the crude was purified by silica gel column chromatography using 5% ethyl acetate in hexane as eluent to obtained aldehyde (A) [7-(4,5-dihexyl-l,3-dioxolan-2-yl) heptanal] (340 g, 43 %) as a colorless oil.

[0431] Preparation of compounds of Formula I-A using 7-(4,5-dihexyl-l,3-dioxolan-2-yl)heptanal [Common A]

[0432]

[0433] Lipid A001

[0434] Stage-6 (Lipid A001); Synthesis of [Nl, Nl-bis(7-(4,5-dihexyl-l,3-dioxolan-2-yl)heptyl)-N2, N2-dim ethylethane- 1,2-diamine]

[0435]

[0436] To a solution of [7-(4,5-dihexyl-l,3-dioxolan-2-yl) heptanal] Common-A (300 mg, 0.0088 mmol, 2.5 equiv) in DCM (10 Vol) was prepared and (1-amino Butanol) Amine-A (26 mg, 0.0029 mmol, 1.0 equiv) was added followed by Sodium tri acetoxy Borohydride (STAB) (561 mg 0.026 mmol, 3.0 equiv ) at 0°C and continuously stirred and allowed to RT for 14 h. The progress was monitored by TLC (using mobile phase;20% EtOAc-n-hexanes) and observed ~25 % of common-A remained intact. Using mobile phase; 10% MeOH-DCM for SM-Amine was absent and Product (I2 active, Ninhydrin anisaldehyde active), the reaction mixture was quenched with ice cold water (5 mb), diluted with DCM (20 Vol) and followed by addition of 10% aq NaHCO3 solution (10 mL) leading to separation of the organic layer. It was again washed with water (10 mL). The finally separated organic layer was dried over Na2SC>4, filtered and MLs were evaporated under reduced pressure resulting in crude residue 1stcolumn purification on silica gel (60-120 mesh) which resulted in major 190 mg of (Lipid A001) & again after 2ndpurification by column using Neutral Al₂O₃, 70 mg of Lipid A001, [N1, N1-bis(7-(4,5-dihexyl-l,3-dioxolan-2-yl)heptyl)-N2, N2-dimethylethane-l,2-diamine] (70 mg, 10%) as a colorless oil was obtained.

[0437] 1HNMR(400 MHz, CDC13) 5: ppm 5.10-4.86 (m, 1H); 4.01-3.89 (m, 2H); 3.56-3.54(m, 2H); 2.59-2.44 (m, 6 H); 1.75-1.21 (m, 31 H); 0.90-0.86(m, 6H).

[0438] Lipid A002

[0439] Stage-6 (Lipid A002): Synthesis of [7-(4,5-dihexyl-l,3-dioxolan-2-yl)-N-(7-(4,5-dihexyl-l,3-dioxolan-2-yl)heptyl)-N-(2-(4-methylpiperazin-l-yl)ethyl)heptan-l-amine]

[0440]

[0441] A solution of [7-(4,5-dihexyl-l,3-dioxolan-2-yl) heptanal] Common-A (300 mg, 0.0088 mmol, 3 equiv) in DCM (10 Vol) was prepared and Amine-B (43 mg, 0.0029 mmol, 1.0 equiv) was added followed by addition of Sodium tri acetoxy Borohydride (STAB) (561 mg 0.026 mmol, 3.0 equiv ) at 0°C and continuously stirred and allowed to RT for 14 h. The progress was monitored by TLC (using mobile phase;20% EtOAc-n-hexanes) and observed ~25 % of Common-A remained intact. Using mobile phase; 10% MeOH-DCM for SM-Amine was absent and Product (I2 active, Ninhydrin anisaldehyde active), the reaction mixture was quenched with ice cold water (5 mL), diluted with DCM(20 Vol) followed by addition of 10% aq NaHCO3 solution(10 mL) leading to separation of the organic layer which was washed again with water (10 mL). The finally separated oiganic layer was dried over Na₂SO₄. It was filtered and MLs were evaporated under reduced pressure resulted crude residue After column purification on silica gel (60-120 mesh) resulted major 190 mg of (Lipid A002) again 2ndpurification by column using Neutral Al₂O₃ resulted pure 150 mg of Lipid A002 followed by treated with activated charcoal in n-pentanes (20 mL) and after filtration and drying 150 mg of Lipid A002, [7-(4,5-dihexyl-l,3-dioxolan-2-yl)-N-(7-(4,5-dihexyl-l,3-dioxolan-2-yl)heptyl)-N-(2-(4-methylpiperazin-l-yl)ethyl)heptan-l-amine] (150 mg, 20.7%) was obtained as a colorless oil.

[0442] 1HNMR(400 MHz, CDC13) 5: ppm 5.10-4.85 (m, 1H); 4.01-3.89 (m, 2H); 3.65-3.49(m, 2H); 2. 94-2.56 (m, 11 H); 2.39-2.34 (m, 3 H); 1.67-1.25 (m, 58 H); 0.90-0.86(m, 12H).

[0443] CAD 89%

[0444] Lipid A003

[0445] Stage-6 (Lipid A003); Synthesis of [4-(bis(7-(4,5-dihexyl-l,3-dioxolan-2-yl)heptyl)amino)butan-l-ol]

[0446]

[0447] A solution of [7-(4,5-dihexyl-l,3-dioxolan-2-yl) heptanal] Common-A (300 mg, 0.0088 mmol, 2.5 equiv) in DCM (10 Vol) was prepared, and (1-amino Butanol) Amine-G (26 mg, 0.0029 mmol, 1.0 equiv) was added, followed by addition of Sodium tri acetoxy Borohydride (STAB) (561 mg 0.026 mmol, 3.0 equiv ) at 0°C and continuously stirred and allowed to RT for 14 h. The progress was monitored by TLC (using mobile phase;20% EtOAc-n-hexanes) and observed that ~25 % of common-A remained intact. Using mobile phase; 10% MeOH-DCM for SM-Amine was absent and Product (h active, Ninhydrin anisaldehyde active), the reaction mixture was quenched with ice cold water (5 mL), diluted with DCM(20 Vol) followed by addition of 10% aq NaHCO3 solution(10 mL), leading to separation of the organic layer which was again washed with water (10 mL) and finally obtained the separated organic layer. It was dried over Na2SC>4, filtered and MLs evaporated under reduced pressure resulting in crude residue. 1stcolumn purification on silica gel (60-120 mesh) resulted in major 260 mg of (Lipid A003). It was followed by treatment with activated charcoal in n-pentanes (20 mL) and after filtration and drying 250 mg of Lipid A003, [4-(bis(7-(4,5-dihexyl-l,3-dioxolan-2- yl)heptyl)ammo)butan-l-ol] (250 mg, 37%) as a colorless oil was obtained.

[0448] ’HNMR^OO MHz, CDC13) 5: ppm 5.09-4.85 (m, 1H); 4.01-3.89 (m, 2H); 3.65-3,49(m, 2H); 2. 77-2.71 (m, 3 H); 2.04 (s, 1H); 1.78-1.25 (m, 50 H); 0.95-0.82(m, 12H).

[0449] EXAMPLE 2: Stagewise process of preparing lipids of Formula 1-B

[0450] Stage-1, (Synthesis of [4-(benzyloxy) butan-l-ol])

[0451] .o.,o.

[0452] Stage-2 Stage-3

[0453] Stage-1

[0454]

[0455] 0HNaH. THF

[0456] BnBr

[0457] Stage-1

[0458]

[0459] First, a 250 mL two-neck round bottom flask was evacuated and backfilled with nitrogen. To this, butane- 1,4-diol 1 (55.48 mmol, 5.0 g, 1.0 equiv) was taken and 100 mL dry THF was added. Then the reaction mixture was cooled down to 0 °C, and to the stirring solution, NaH (60% dispersion in mineral oil, 55.48 mmol, 1.33 g, 1.0 equiv) was added in portion under positive nitrogen pressure. The resulting mixture was stirred at room temperature for 30 min and again cooled down to 0 °C. After that, tetrabutylammonium iodide (TBAI, 5.54 mmol, 2.0 g, 0.1 equiv) was added followed by benzyl bromide (44.38 mmol, 7.5 gm, 0.8 equiv) was added for 15 min and the resulting solution was stirred at room temperature for 2 hrs. After complete consumption of A, as indicated by TLC, the reaction mixture was quenched with water (60 mL) and the aqueous layer was extracted with ethyl acetate (3 x 60 mL). The combined organic layers were dried over anhydrous Na₂SO₄ and evaporated under reduced pressure. The crude residue was purified by silica gel column chromatography using 35% ethyl acetate in hexane as eluent to afford Int-2, [4-(benzyloxy) butan-l-ol] (5.0 g, 50 %), respectively, as pale-yellow oil.1HNMR(400 MHz, CDC13) 5: ppm 7.37-7.26 (m, 5H); 4.50(s, 2H);.3.71-3.64 (m, 2H); 3.54-3.49 (m,2H); 1.75-1.63 (m, 4H).

[0460] Stage-2: Synthesis of [ 4-(benzyloxy) butanal]

[0461] . OBn PCC. OBn

[0462] HO' O'

[0463] 2 Stage-2 3

[0464] Chemical Formula: C11H16O2Chemical Formula: C₁₁H₁₄O₂

[0465] Exact Mass: 180.12 Exact Mass: 178.10

[0466]

[0467] Molecular Weight: 180.25 Molecular Weight: 178.23

[0468] A 500 mL two-neck round bottom flask was evacuated and backfilled with nitrogen, and to this 150 ml of MDC was added once again and nitrogen purged thoroughly. Then the pyridinium chlorochromate (PCC) (23.8 g, 110.49 mmol, 1.2 equiv) was added to above solution and stirred for 5min. To the above reaction mixture, solution of 4-(benzyloxy)-l -butanol (2) (18.8 g, 103.22 mmol, 1 equiv) in dichloromethane (30 mL) was added slowly for 15 to 20 min c. The reaction was stirred at room temp, for 2 to 3 hrs, after completion of reaction, reaction mixture was filtered through hyflow to remove the black insoluble material, eluting with dichloromethane. After evaporation of the filtrate, the crude was purified by silica gel column chromatography using 5% ethyl acetate in hexane as eluent to obtain aldehyde (3) [ 4-(benzyloxy) butanal] (12.0 g, 64%) as a colorless oil.

[0469] 1HNMR (CDC13): *HNMR (400 MHz, CDC13) 5: ppm 9.79-9.78 (t, 1H, J=2.8); 7.37-7.28 (m, 5H); 4.50-4.49 (s, 2H);.3.52-3.47 (m, 2H); 2.57-2.53 (m,2H); 1.98-1.92 (m, 2H).

[0470] Stage-3: Synthesis of [((4,4-dimethoxybutoxy) methyl) benzene]

[0471]

[0472] A stirred solution of comp-3 (10.0 gm, 178.10 mmol, 1 equiv) in methanol (100 ml), and trimethyl orthoformate (17.7 gm, 106.12 mmol, 3 equiv) was added followed by PTSA (200 mg, 20 mol %) at RT. The reaction mixture stirred for overnight at r.t. The resulting mixture was washed with 5% NaOH aq. Solution (20 ml) and extracted with EtOAC (2x100 ml). The organic layer was separated, dried (Na₂SO₄) and evaporated to give crude compound, which was purified using silicagel column chromatography using 5% ethyl acetate in hexane as eluent to obtain acetal-(4) [ ((4,4-dimethoxybutoxy) methyl) benzene] (8.1 g, 64.4%) as a colorless oil.

[0473] ¹H NMR (400 MHz, CDC13) 5: ppm 7.34-7.26 (m, 5H); 4.50 (s, 2H); 4.39-4.37(t, 1H, J = 11 Hz).3.50-3.47 (t, 2H, J= 9.6 Hz); 3.34 (s, 6H); 1.72-1.63 (m, 4H).

[0474] Stage-4: Synthesis of [ tetradecane-7, 8-diol]:

[0475] Synthesis for common lnt-9

[0476]

[0477]

[0478] A mixture of 7-tetradecene (purchased from TCI.) (7.9 g, 40.22 mmol), 7.1 ml of 37% H2O2 and 49 ml of formic acid was stirred at the temperature 40-50° C for 7 hrs. The reaction mixture was concentrated under a rotary evaporator in a hot water bath at ~30 torr to remove most of the water and formic acid. The above crude was washed with water (10 Vol) and extracted with ethyl acetate (15Vol) followed by concentration on a rotary evaporator. Then, residue dissolved in 10% KOH in Methanol (50 ml) and 100 ml toluene and heated to reflux for 6 hrs. The reaction mixture concentrated and residue dissolved in water and diluted with 500 mL ethyl acetate and transferred to a separatory funnel. The organic layer was separated, and the aqueous layer was extracted 3 times (3x200 mL) with ethyl acetate. The ethyl acetate extracts were combined and dried over anhydrous Na₂SO₄ Filtration, followed by concentration on a rotary evaporator at reduced pressure. The above crude was purified using silica gel column chromatography using 12% ethyl acetate in hexane as eluent to obtain desired diol-(6) [ tetradecane-7, 8-diol] as white powder (7.1 g, 77%).

[0479] 'H NMR (400 MHz, CDC13) 5: ppm 4.98-4.89 (m, 1H); 3.72-3.68(m, 1H); 1.68-1.26 (m, 20 H); 0.87-0.81(m, 6H).

[0480] Stage-5: Synthesis of [ 2-(3-(benzyloxy) propyl)-4,5-dihexyl-l,3-dioxolane]:

[0481]

[0482] A solution of diol-6 (2.55gm, 11.07mmol, 1 equiv), comp-4 (2.97 gm, 13.29 mmol, 1.2 eqiv) in toluene (30 ml) was added PTSA (310 mg, 0.1 equi) and the reaction mixture heated under reflux 90 to 100° C for overnight. After completion of the reaction cool the reaction mixture and evaporated, the above crude was purified using silicagel column chromatography using 2% ethyl acetate in hexane as eluent to obtain Int-(7) [ 2 -(3 -(benzyloxy) propyl)-4, 5 -dihexyl -1,3 -dioxolane] as colorless oil (3.74 g, 86.5 %).

[0483] ’HNMR (400 MHz, CDC13) 5: ppm 7.33-7.24 (m,5 H); 5.14-4.90 (m, 1H); 4.50 (s, 2H); 4.01-3.89 (m, 2H); 3.55-3.48(m, 2H); 1.77-1.29 (m, 24 H); 0.90-0.86(m, 6H).

[0484] Stage-6: Synthesis of [3-(4,5-dihexyl-l,3-dioxolan-2-yl) propan-l-ol

[0485]

[0486] A solution of [ 2-(3 -(benzyloxy) propyl)-4, 5 -dihexyl- 1,3 -dioxolane] Int-7 (3.5 gm, 8.96 mmol, 1.0 equiv.) in Ethyl acetate (30 mL) was made and Pd / C (30 mg, 0.04 mmol, 0.1 equiv.) was added. The reaction mixture was purged with H2 gas and stirred under an atmosphere of H2 (applied through balloon filled with hydrogen gas) for 12 h. After complete consumption of starting material as indicated by TLC, the reaction mixture was filtered through a pad of celite and washed with ethyl acetate (100 mL). The filtrates were then concentrated under reduced pressure and purified by silica gel column chromatography using 18% ethyl acetate in n-hexane as eluent to afford Int-8, [3-(4,5-dihexyl-l,3-dioxolan-2-yl) propan-l-ol] (2.6 gm, 96%) as a colorless oil.

[0487] 1HNMR(400 MHz, CDC13) 5: ppm 5.15-4.92 (m, 1H); 4.06-3.93 (m, 2H); 3.69-3.49(m, 2H); 1.95-1.25 (m, 24 H); 0.90-0.79(m, 6H).

[0488] Stage-7: Synthesis of [3-(4,5-dihexyl-l,3-dioxolan-2-yl) propyl acrylate]

[0489]

[0490] A 50 mL two-neck round bottom flask was evacuated and backfilled with nitrogen, to this 5 ml of MDC was added once again nitrogen purged thoroughly. Then the Int-8 (1.0 gm, 3.33 mmol, 1.0 equiv) was added and the reaction mixture was cooled down to 0° C. To this DIPEA (2.5 gm, 19.28 mmol, 6.0 equiv) and acryloyl chloride (0.67 ml, 8.32 mmol, 2.5 equiv) were added slowly under positive nitrogen pressure. The resulting mixture was stirred at room temperature for 6 hrs. The progress of the reaction was checked by TLC and showed absence of SM. Reaction mixture was diluted with water and extracted MDC (50 mL x 2). The combined organic layer was dried over anhydrous Na₂SO₄, evaporated under vacuo to get crude. Crude compound was purified by silica gel column chromatography using 5% ethyl acetate in hexane as eluent to afford Common-9, [3-(4,5-dihexyl-l,3-dioxolan-2-yl) propyl acrylate] (970 mg, 82.2%) as a colorless oil.

[0491] ‘HNMR (400 MHz, CDC13) 5: ppm 6.42-6.37 (dd, 1H, J = 16 Hz); 6.14-6.08 (m, 1H); 5.82-5.80 (dd, 1H, J=8Hz); 5.15-4.92 (m, 1H); 4.22-4.17 (m, 2H); 3.93-3.90(m, 1H); 3.56-3.55 (m,1 H), 1.83-1.29 (m, 24 H); 0.90-0.87(m, 6H).

[0492] Lipid B001

[0493] Lipid B002

[0494]

[0495] Lipid B006 Lipid B003

[0496] Lipid B001

[0497] Stage-8 (Lipid B001); Synthesis of [bis(3-(4,5-dihexyl-l,3-dioxolan-2-yl) propyl) 3,3'-((2-(dimethylamino)ethyl) azanediyl) dipropionate]

[0498]

[0499] A solution of Common-9 (300 mg, 0.0084 mmol, 3 equiv) in Ethanol (3 Vol) was prepared and Amine-A (25 mg, 0.0028 mmol, 1 eq,) added in two portions with 3hr intervals, at 60°C, for 6 h. The progress was monitored by TLC (using mobile phase;20% EtOAc-n-hexanes) and observed that ~30 % of common-9 remained intact. Using mobile phase; 10% MeOH-DCM for SM-Amine was absent and Product (I₂ active) along with undesired alcohol of 9 was observed, the reaction mixture was under reduced pressure resulting in a crude residue. After column purification on silica gel (60-120 mesh) resulted in major 170 mg mixture of (Lipid BOO 1 and undesired alcohol of common-9, 1: 1 ratio). 2ndpurification by column using Neutral Al₂O₃ resulted pure 100 mg of Lipid B001 followed by treatment with activated charcoal in n-pentanes (20 mL) and subsequent filtration and drying, 90 mg of Lipid B001, [bis(3-(4,5-dihexyl-l,3-dioxolan-2-yl) propyl) 3,3'-((2-(dimethylamino)ethyl) azanediyl) dipropionate] (90 mg, 13%) was obtained as a colorless oil.

[0500] 1HNMR(400 MHz, CDC13) 5: ppm 5.12-4.89 (m, 1H); 4.10-4.07 (m, 2H); 4.02-3.90 (m, 2H); 3.55 (m,1H); 3.16-3.15(m, 1H); 2.99 (m, 1H); 2.82 (s, 3 H);2.78-2.74 (m, 2H)2.49-2.46 (m, 2H); 1.77-1.25 (m, 42 H); 0.89-0.82(m, 9H).

[0501] Lipid B002

[0502] Stage-8 (Lipid B002): Synthesis of bis(3-(4,5-dihexyl-l,3-dioxolan-2-yl) propyl) 3,3'-((2-(4-methylpiperazin-l-yl) ethyl) azanediyl) dipropionate

[0503]

[0504] A solution of Common-9 (300 mg, 0.0084 mmol, 3 equiv) in Ethanol (3 Vol) was prepared and Amine-B (40 mg, 0.0028 mmol, 1 eq,) was added in two portions for 3hr intervals), at 60°C, for 6 h. The progress was monitored by TLC (using mobile phase;20% EtOAc-n-hexanes) and observed that ~30 % of common-9 remained intact. Using mobile phase; 10% MeOH-DCM for SM-Amine was absent and Product (I₂ active) along with undesired alcohol of 9 was observed, the reaction mixture was under reduced pressure resulted crude residue After column purification on silica gel (60-120 mesh) resulted in major 170 mg mixture of (Lipid B002 and undesired alcohol of common-9, 1:1 ratio ) and again 2ndpurification by column using Neutral Al₂O₃ resulted in pure 100 mg of Target-002 followed by treatment with activated charcoal in n-pentanes (20 mL) and subsequent filtration and drying, 24 mg of Lipid B002, bis(3-(4,5-dihexyl-l,3-dioxolan-2-yl) propyl) 3,3'-((2-(4-methylpiperazin-l-yl) ethyl) azanediyl) dipropionate (24 mg,) was obtained as a colorless oil. ’HNMR^OO MHz, CDC13) 5: ppm 5.12-4.89 (m, 2H); 4.10-3.90 (m, 1H); 3.40-3.37 (m,4H); 2.80-2.76 (m, 8 H); 2.62 (m, 3H); 2.47-2.36 (m, 11H); 2.08-2.00 (m, 4 H); 1.75-1.71 (m, 8 H); 1.48-1.45 (m, 10 H); 1.40-1.29 (m, 31 H); 0.90-0.87(m, 12H).

[0505] LCMS-Q TOF: 91% pure m / z: 852.7

[0506] Lipid B003

[0507] Stage-8 (Lipid B003): Synthesis of [bis(3-(4,5-dihexyl-l,3-dioxolan-2-yl) propyl) 3,3'-((2-(l-methylpyrrolidin-2-yl) ethyl) azanediyl) dipropionate]

[0508]

[0509] A solution of Common-9 (300 mg, 0.0084 mmol, 3 equiv) in Ethanol (3 Vol) was prepared and Amine-D (36 mg, 0.0028 mmol, 1 eq,) was added in two portions for 3hr intervals), at 60°C, for 6 h. The progress was monitored by TLC (using mobile phase;20% EtOAc - n-hexanes) and observed that ~30 % of common-9 remained intact. Using mobile phase;10% MeOH-DCM for SM-Amine was absent and Product (I₂ active) along with undesired alcohol of 9 was observed, the reaction mixture was under reduced pressure resulting in crude residue. After column purification on silica gel (60-120 mesh) resulted in major 150 mg mixture of (Lipid B003 and undesired alcohol of common-9, 3:1 ratio ) and again 2ndpurification by column using Neutral Al₂O₃ resulted in 120 mg of Lipid B003 followed by treatment with activated charcoal in n-pentanes (20 mL) and subsequent filtration and drying 118 mg of Lipid B003, [bis(3-(4,5-dihexyl-l,3-dioxolan-2-yl) propyl) 3,3'-((2-(l-methylpyrrolidin-2-yl) ethyl) azanediyl) dipropionate] (118 mg, 17%) was obtained as a colorless oil.

[0510] 1HNMR(400 MHz, CDC13) 5: ppm 5.14-4.89 (m, 1H); 4.12-3.88 (m, 3H); 3.57-3.49 (m, 1H); 2.87-2.39 (m,9H); 2.22-1.25(m, 27H); 0.97-0.82(m, 6H).

[0511] Lipid B004 Stage-8, (Lipid B004): Synthesis of [bis(3-(4,5-dihexyl-l,3-dioxolan-2-yl) propyl) 3,3'-((2-(4-hydroxypiperidin-l-yl) ethyl) azanediyl) dipropionate]

[0512]

[0513] A solution of Common-9 (300 mg, 0.0084 mmol, 3 equiv) in Ethanol (3 Vol) was prepared and Amine-C (40 mg, 0.0028 mmol, 1 eq,) was added in two portions for 3hr intervals, at 60°C, for 6 h. The progress was monitored by TLC (using mobile phase;20% EtOAc-n-hexanes) and observed that ~25 % of common-9 remained intact. Using mobile phase; 10% MeOH-DCM for SM- Amine was absent and Product (I₂ active) along with undesired alcohol of 9 was observed, the reaction mixture was under reduced pressure resulted crude residue After column purification on silica gel (60-120 mesh) resulted in major 160 mg mixture of (Lipid B004 and undesired alcohol of common-9, 3:1 ratio ) and again 2ndpurification by column using Neutral Al₂O₃ resulted in 150 mg of Target-04, followed by treatment with activated charcoal in n-pentanes (20 mL) and subsequent filtration and drying, 123 mg of Lipid B004, [bis(3-(4,5-dihexyl-l,3-dioxolan-2-yl) propyl) 3,3'-((2-(4-hydroxypiperidin-l-yl) ethyl) azanediyl) dipropionate] (123 mg, 17%) was obtained as a colorless oil.

[0514] 1HNMR(400 MHz, CDC13) 5: ppm 5.12-4.90 (m, 1H); 4.10-3.90 (m, 4H); 3.56 (m, 1H); 3.27-2.74 (m, 8H); 2.48-1.25 (m, 50 H); 0.95-0.87(m, 9H).

[0515] Lipid B005

[0516] Stage-8, (Lipid B005): Synthesis of [bis(3-(4,5-dihexyl-l,3-dioxolan-2-yl) propyl) 3,3'-((4-(4-methylpiperazin-l-yl) butyl) azanediyl) dipropionate]

[0517]

[0518] A solution of Common-9 (300 mg, 0.0084 mmol, 3 equiv) in Ethanol (3 Vol) was prepared and Amine-E (40 mg, 0.0028 mmol, 1 eq,) was added in two portions for 3hr intervals, at 60°C, for 6 h. The progress was monitored by TLC (using mobile phase;20% EtOAc-n-hexanes) and observed that ~25 % of common-9 remained intact. Using mobile phase; 10% MeOH-DCM for SM-Amine was absent and Product (I₂ active) along with undesired alcohol of 9 was observed, the reaction mixture was under reduced pressure resulted crude residue. After column purification on silica gel (60-120 mesh) resulted major 240 mg mixture of (Lipid B005 and undesired alcohol of common-9, 3:1 ratio ) and again 2ndpurification by column using Neutral Al₂O₃ resulted 65 mg of Target-005 followed by treatment with activated charcoal in n-pentanes (20 mL) and after filtration and drying 58 mg (Lipid B005) was obtained which was dissolved in DCM (20 mL)and given water wash (5mL) separated organic layer which was dried over Na₂SO₄ and then filtered MLs were evaporated under reduced pressure. 40 mg of Lipid B005, [bis(3-(4,5-dihexyl-l,3-dioxolan-2-yl) propyl) 3,3'-((4-(4-methylpiperazin-l-yl) butyl) azanediyl) dipropionate] (50 mg, 5.5 %) was obtained as a colorless oil.

[0519] 'H NMR (400 MHz, CDC13) 5: ppm 5.12-4.90 (m, 1H); 4.10-4.08 (m, 1H); 3.56 (m, 1H); 2.75(t, 1 H); 2.44-2.29 (m, 5 H); 1.75-1.25 (m, 35 H); 0.88-0.83(m, 6H).

[0520] Lipid B006

[0521] Stage-8, (Lipid B006): Synthesis of [bis(3-(4,5-dihexyl-l,3-dioxolan-2-yl) propyl) 3,3'-((4-(4-hydroxypiperidin-l-yl) butyl) azanediyl) dipropionate]

[0522]

[0523] A solution of Common-9 (300 mg, 0.0084 mmol, 3 equiv) in Ethanol (3 Vol) was prepared and Amine-F (48 mg, 0.0028 mmol, 1 eq,) was added in two portions for 3hr intervals, at 60°C, for 6 h. The progress of the was monitored by TLC (using mobile phase;20% EtOAc-n-hexanes) and observed that ~25 % of common-9 remained intact. Using mobile phase;10% MeOH-DCM for SM-Amine was absent and Product (I₂ active) along with undesired alcohol of 9 was observed, the reaction mixture was under reduced pressure resulting in crude residue. After column purification on silica gel (60-120 mesh) resulted in major 160 mg mixture of (Lipid B006 and undesired alcohol of common-9, 3:1 ratio ) and again 2ndpurification by column using Neutral Al₂O₃ resulted in 195 mg of Lipid B006 followed by treatment with activated charcoal in n-pentanes (20 mL) and subsequent filtration and drying, 162 mg of Lipid B006, [bis(3-(4,5-dihexyl-l,3-dioxolan-2-yl) propyl) 3,3'-((4-(4-hydroxypiperidin-l-yl) butyl) azanediyl) dipropionate] (162 mg, 17%) was obtained as a colorless oil.

[0524] 1HNMR(400 MHz, CDC13) 5: ppm 5.14-4.89 (m, 1H); 4.12-3.90 (m, 3H); 3.56 (m, 1H); 3.07-2.74(m,13 H); 1.84-1.25 (m, 24 H); 0.90-0.85(m, 6H).

[0525] EXAMPLE 3: Stagewise process of preparing lipids of Formula 1-M

[0526]

[0527] Stage 1: Synthesis of [3-(4,5-dihexyl-l,3-dioxolan-2-yl) propyl methanesulfonate]

[0528] A 50 mL two-neck round bottom flask was evacuated and backfilled with nitrogen, to this 50 ml of MDC was added once again and purged with thoroughly. Then the Int-7 (2 gm, 6.6 mmol, 1.0 equiv) was added and the reaction mixture was cooled down to 0° C. To this TEA (2.8 mL, 19.9 mmol, 3.0 equiv) and Methane sulphonyl chloride (0.8 ml, 9.9 mmol, 1.5 equiv) were added slowly under nitrogen atmosphere. The resulting mixture was stirred at 0°C for 2 hrs. The progress of the reaction was monitored by TLC and showed absence of SM. The reaction mixture was diluted with water and MDC (50 mL x 2) was extracted. The combined organic layer was dried over anhydrous Na2SC>4, evaporated under vacuo to get crude compound Int-8, [3-(4,5-dihexyl-l,3-dioxolan-2-yl) propyl methanesulfonate ] (2.5 gm, 100 % yield) as a colorless oil, directly utilizing for next step.

[0529] Stage 2: Synthesis of [N-benzyl-3-(4,5-dihexyl-l,3-dioxolan-2-yl) propan-l-amine] A solution of Int-8 (2.5 gm, 6.6 mmol, 1.0 equiv) and benzyl amine (12 mL neat) was prepared and stirred. The resulting mixture was stirred at 110°C for 14 h under sealed tube, The progress of the reaction was monitored by TLC and showed absence of SM. Reaction mixture diluted with water and EtOAc (50 mL x 2)was extracted. The organic layer was washed with 10% aq NaHSO₄ wash (2x15 ml). The combined organic layer was dried over anhydrous Na2SC>4, evaporated under vacuo to get crude. This was purified using Basic Al₂O₃ (eluted at 3% ethyl acetate-hexane) to obtain Int-9, [N-benzyl-3-(4,5-dihexyl-l,3-dioxolan-2-yl) propan-l-amine] (1.5gm, 58 % yield) as a colorless oil.

[0530] Stage 3: Synthesis of [3-(4,5-dihexyl-l,3-dioxolan-2-yl)propan-l-amine

[0531] A solution of [ 2-(3 -(benzyloxy) propyl)-4, 5 -dihexyl- 1,3 -dioxolane] Int-7 (1.5 gm, 3.85 mmol, 1.0 equiv) in MeOH (50 mL) was prepared and 10% Pd / C-50% wet in H2O (350 mg) was added. The reaction mixture was kept under H2 gas pressure 100 Psi at 55C for 12 h. After complete consumption of starting material as indicated by TLC, the reaction mixture was filtered through a pad of celite, and washed with MeOH (20 mL). The filtrates were then concentrated under reduced to afford common-6, [3-(4,5-dihexyl-l,3-dioxolan-2-yl)propan-l-amine] (1.1 gm, 95.6 % yield) as a colorless oil.

[0532] Lipid M001

[0533]

[0534] Stage 4: Synthesis of [dimethyl 3,3'-((2-(4-methylpiperazin-yl)ethyl)azanediyl)dipropionate] A solution of 2-(4-methylpiperazin-l-yl)ethan-l -amine B-73 (500 mg, 3.50 mmol, 1.0 equiv) in MeOH (10 mL) was prepared and Methyl acrylate (1.5 gm, 17.4 mmol, 5.0 equiv) was added at RT resulting in a reaction mixture at 60°C for 12 h, in a sealed tube After complete consumption of starting material as indicated by TLC, the reaction mixture was concentrated under reduced to afford Int-A, [dimethyl 3,3'-((2-(4-methylpiperazin-l-yl)ethyl)azanediyl)dipropionate] (1.1 gm, 100% yield) as a colorless oil.

[0535] Stage 5: Synthesis of [3,3'-((2-(4-methylpiperazin-l-yl)ethyl)azanediyl)dipropionic acid] A solution of Int-A, [dimethyl 3,3'-((2-(4-methylpiperazin-l-yl)ethyl)azanediyl)dipropionate (1.2 gm, 3.80 mmol, 1.0 equiv) in 1,4-dioxane (20 mL) was prepared and TFA:6N Aq HC1(6 mL, 1:1, 5 vol) was added, at RT, resulting in a reaction mixture at 80°C for 4 h. After complete consumption of starting material as indicated by TLC, the reaction mixture was concentrated under reduced to afford Int-B, [3,3'-((2-(4-methylpiperazin-l-yl)ethyl)azanediyl)dipropionic acid] (1.3 gm, 85.5% (TFA salt), yield) as a low melting solid.

[0536] Stage 6: Synthesis of [3,3'-((2-(4-methylpiperazin-l-yl)ethyl)azanediyl)bis(N-(3-(4,5-dihexyl-l,3-dioxolan-2-yl)propyl)propanamide)]

[0537] A solution of diacid- Int-B (320 mg, 2.00 mmol, 1 equiv) in DCM (10 mL) was stirred and the resulting reaction mixture was cooled down to 0° C, under nitrogen atmosphere and EDCI (600 mg 8.00 mmol, 4 eq), DIPEA (0.83 mL, 12.00 mmol, 6.0 equiv) was added along with Common-Int-6 (600 mg, 2.00 mmol, 1.0 equiv). The resulting mixture was stirred at room temperature for 12 hrs. The progress of the reaction was checked by TLC and showed absence of SM. Reaction mixture was diluted with water and extracted EtOAc (30 mL). The organic layer washed with 10% aq NaHCO3 wash. The combined organic layer was dried over anhydrous Na2SC>4, evaporated under vacuo and crude compound was obtained, which was purified using basic Al₂O₃ (eluted at 2% MeOH in DCM) resulting in obtaining Target-8, [3,3'-((2-(4-methylpiperazin-l-yl)ethyl)azanediyl)bis(N-(3-(4,5-dihexyl-l,3-dioxolan-2-yl)propyl)propanamide)] (270 mg, 16% yield) as a pale yellow oil.

[0538]

[0539] Lipid M002

[0540]

[0541] Stage 4: Synthesis of [dimethyl 3,3'-((2-(dimethylamino)ethyl)azanediyl)dipropionate] A solution of N1, N1 -dimethylethane- 1,2-diamine (SM-1)(1 gm, 11.30 mmol, 1.0 equiv) in MeOH (10 mL) was prepared and added Methyl acrylate (SM-2) (3.9 gm, 45.4 mmol, 4.0 equiv) was added at RT, resulting in a reaction mixture at 60°C for 12 h, in a sealed tube. After complete consumption of starting material as indicated by TLC, the reaction mixture was concentrated under reduced to afford Int-C, [dimethyl 3,3'-((2-(dimethylamino)ethyl)azanediyl)dipropionate] (2.5 gm, 85% yield) as a colorless oil.

[0542] Stage 5: Synthesis of [3,3'-((2-(4-methylpiperazin-l-yl)ethyl)azanediyl)dipropionic acid] A solution of Int-c, [dimethyl 3,3'-((2-(4-methylpiperazin-l-yl)ethyl)azanediyl)dipropionate (1 gm, 3.84 mmol, 1.0 equiv) in 1,4-dioxane (10 mL) was prepared and TFA:6N Aq HC1( 5 mL, 1: 1, 5 vol) was added, at RT, resulting in a reaction mixture at 80°C for 4 h. After complete consumption of starting material as indicated by TLC, the reaction mixture was concentrated under reduced to afford Int-D, [3,3'-((2-(4-methylpiperazin-l-yl)ethyl)azanediyl)dipropionic acid] (950 mg, 71% (TFA salt) yield) as a low melting solid.

[0543] Stage 6: Synthesis of [3,3'-((2-(dimethylamino)ethyl)azanediyl)bis(N-(3-(4,5-dihexyl-l,3-dioxolan-2-yl)propyl)propanamide)]

[0544] A solution of diacid-Int-D (310 mg, 2.00 mmol, 1 equiv ) in DCM (10 mL) was stirred resulting in a reaction mixture which was cooled down to 0° C. under nitrogen atmosphere. EDCI (682 mg 8.00 mmol, 4 eq), DIPEA (0.9 mL, 12.00 mmol, 6.0 equiv) and common-Int-6 (600 mg, 2.00 mmol, 1.0 equiv) were added. The resulting mixture was stirred at room temperature for 12 hrs. The progress of the reaction was checked by TLC and showed absence of SM. Reaction mixture diluted with water and extracted EtOAc (30 mL). The organic layer was washed with 10% aq NaHSO₄ followed by 5% aq NaHCO₃ wash ( The combined organic layer was dried over anhydrous Na2SC>4, evaporated under vacuo to obtain crude compound which was purified using basic Al₂O₃ (eluted at 2% MeOH in DCM) to afford Target-9, [3,3'-((2- (dimethylamino)ethyl)azanediyl)bis(N-(3 -(4,5 -dihexyl- 1,3 -dioxolan-2-yl)propyl)propanamide)] (200 mg, 12.6% yield) as a pale yellow oil.

[0545]

[0546] EXAMPLE 4: Stagewise process of preparing lipids of Formula 1-D

[0547] Stage 1: Synthesis of [6-(benzyloxy)hexan-l-ol] - Lipid D003

[0548] A solution of hexane- 1,6-diol (3 gm, 25.40 mmol, 1.0 equiv) was taken and 100 mL dry THF was added. Then the reaction mixture was cooled down to 0 °C, and to the stirring solution, NaH (60% dispersion in mineral oil, 1.0 gm, 25.40 mmol, 1.0 equiv) was added in portion under positive nitrogen pressure. The resulting mixture was stirred at room temperature for 30 min and again cooled down to 0 °C. After that, tetrabutylammonium iodide TBAI, 938 mg, 2.54 mmol, 0.1 equiv) was added followed by Benzyl bromide (2.4 mL, 20,00 mmol 0.8 equiv) for 15 min and the resulting solution was stirred at room temperature for 18 hrs. After complete consumption of SM-2, as indicated by TLC, the reaction mixture was quenched with ice cold water (60 mL) and the aqueous layer was extracted with ethyl acetate (3 x 60 mL). The combined organic layers were dried over anhydrous Na2SC>4 and evaporated under reduced pressure. The crude residue was purified by silica gel 60-120 column chromatography using 1% MeOH-DCM as eluent to afford compound 5, [4-(benzyloxy) butan-l-ol] (2.7 gm, 51 % yield), as colorless oil. Directly utilized for next step.

[0549] Stage 2: Synthesis of [2-(4,5-dihexyl-l,3-dioxolan-2-yl) acetic acid]

[0550] A solution of methyl 2-(4,5-dihexyl-l,3-dioxolan-2-yl) acetate Int-3 (3.9 gm, 11.07mmol, 1 equiv), LiOH.H₂O (1.04 gm, 13.29 mmol, 1.2 equiv) in THF: H2O(48 mL:24 ml, 2: 1 ratio) was prepared and the resulting reaction mixture was stirred at RT for overnight. RM was monitored by TLC (10% EA-Hexane), SM was absent, the reaction mixture was diluted with water (50 mL) and acidify using 10% aq naHSO4 and the aqueous layer was extracted with ethyl acetate (3 x 25 mL). The combined organic layers were dried over anhydrous Na₂SO₄ and evaporated under reduced pressure to obtain Int-(4) [ 2-(4,5-dihexyl-l,3-dioxolan-2-yl)acetic acid] as colorless oil (3.6 gm, 97 % yield). Stage 3: Synthesis of [6-(benzyloxy)hexyl 2-(4,5-dihexyl-l,3-dioxolan-2-yl)acetate] A solution of 2-(4,5-dihexyl-l,3-dioxolan-2-yl)acetic acid (Int-4) (2.4 gm, 8.00 mmol, 1 equiv ) in DCM (50 mL) was stirred and the resulting reaction mixture was cooled down to 0° C under nitrogen atmosphere. EDCI (1.9 gm, 9.80 mmol, 1.25 eq), DIPEA (3.4 mL, 20.00 mmol, 2.5 equiv), DMAP (97 mg, 0.80 mmol, 0.1 equiv) and Int-5 (1.7 gm, 8.00 mmol, 1.0 equiv) were added. The resulting mixture was stirred at room temperature for 12 hrs. The progress of the reaction was checked by TLC and showed absence of SM. Reaction mixture was diluted with water and extracted EtOAc (30 mL), The combined organic layer was dried over anhydrous Na2SC>4, evaporated under vacuo was obtained crude, which was purified using silica gel 60-120 mesh (Eluted at 2% EA In Hexane, PMA active) was afford (Int-6) [6-(benzyloxy) hexyl 2-(4,5-dihexyl-l,3-dioxolan-2-yl) acetate] (2.3 gm, 58 % yield) as a color less oil.

[0551] Stage 4: Synthesis of [6-hydroxyhexyl 2-(4,5-dihexyl-l,3-dioxolan-2-yl)acetate]

[0552] A solution of [ 6-(benzyloxy) hexyl 2-(4,5-dihexyl-l,3-dioxolan-2-yl) acetate] Int-6 (4.5 gm, 8.96 mmol, 1.0 equiv) in MeOH (90mL) was prepared and Pd / C (750 mg) was added. The reaction mixture was purged with H2 gas pressure 100 Psi at RT for 14 h. After complete consumption of starting material as indicated by TLC (20% EA-Hexane, PMA active), the reaction mixture was filtered through a pad of celite, and washed with MeOH (100 mL). The filtrates were then concentrated under reduced to afford Int-7, [6-hydroxyhexyl 2-(4,5-dihexyl-l,3-dioxolan-2-yl) acetate] (3.3 gm, 91% yield), as a colorless oil. Directly utilized for next step.

[0553] Stage 5: Synthesis of [6-oxohexyl 2-(4,5-dihexyl-l,3-dioxolan-2-yl)acetate]

[0554] A solution of 6-hydroxyhexyl 2-(4,5-dihexyl-l,3-dioxolan-2-yl)acetate (Int-7) (400 mg, 1 mmol, 1 equiv) in DCM (24 mL) was prepared and cooled to 0°c Then pyridinium chlorochromate (PCC) (260 mg, 1.30 mmol, 1.3 equiv) was added. The reaction was stirred at room temp, for 5 h, after completion of reaction, the reaction mixture was filtered through hyflow bed to remove the black insoluble material, washed with DCM (10 mL). MLs were evaporated obtained crude was purified by silica gel column chromatography using silica gel 60-120 mesh (5% ethyl acetate in hexane) as eluent to obtained Int-(8) [6-oxohexyl 2-(4,5-dihexyl-l,3-dioxolan-2-yl) acetate (280 mg, 70% yield) as a colorless oil.

[0555]

[0556] Stage 6: Synthesis of ((4-hydroxybutyl)azanediyl)bis(hexane-6,l-diyl) bis(2-(4,5-dihexyl-1,3-dioxolan-2-yl)acetate)

[0557] A solution of [6-oxohexyl 2-(4,5-dihexyl-l,3-dioxolan-2-yl)acetate] Int-8 (280 mg, 0.7 mmol, 3 equiv) in DCM (10 m ) and 4-amino butanol (Amine-D) (24 mg, 0.23 mmol, 1.0 equiv) was prepared followed addition of sodium tri acetoxy Borohydride (STAB) (372 mg, 1.7 mmol, 2.5 equiv ) at 0°C and continuously stirred and allowed to RT for 5 h. The progress was monitored by TLC (using mobile phase;20% EtOAc-n-hexanes) and observed that~25 % of Int-8 remained intact. Using mobile phase;10% MeOH-DCM for SM-Amine was absent and Product (L active, PMA-active), the reaction mixture was quenched with ice cold water (5 m ), diluted with DCM(20 Vol) and followed by addition of 10% aq NaHCO₃ solution(10 mL) which separated the organic layer. This was again washed with water (10 mL) and the finally separated organic layer was dried over Na2SC>4, filtered and MLs evaporated under reduced pressure resulting in crude residue purification by column using Basic Al₂O₃. This resulted (eluted at 40% EA -hexane) Lipid D006 [((4-hydroxybutyl)azanediyl)bis(hexane-6,l-diyl) bis(2-(4,5-dihexyl-l,3-dioxolan-2-yl)acetate)] (27 mg, 5 % yield ) as a colorless oil.

[0558] The inventive LNP formulations along with their composition is provided in the Table- 14 below.

[0559] TABLE- 14

[0560] LNP Composition

[0561] Fl B002 (46.3%); DSPC (9.4%); Choi (42.7%); ALC0159 (1.6%) F2 B003 (46.3%); DSPC (9.4%); Choi (42.7%); ALC0159 (1.6%) F3 D003 (46.3%); DSPC (9.4%); Choi (42.7%); ALC0159 (1.6%) F4 A003 (46.3%); DSPC (9.4%); Choi (42.7%); DSPE-PEG2000 (1.6%) F5 A003 (46.3%); DSPC (9.4%); Choi (42.7%); ALC0159 (1.6%)

[0562]

[0563] F6 A003 (41.67%); Lipid 1 from Patent 1 (4.63%); DSPC (9.4%); Choi (42.7%); ALC0159 (1.6%)

[0564] F7 A003 (41.67%); Lipid 1 (4.63%); DSPC (9.4%); Choi (42.7%);

[0565] DSPE-PEG2000 (1.6%)

[0566] F9 A009 (46.3%); DSPC (9.4%); Choi (42.7%); ALC0159 (1.6%) F10 A015 (46.3%); DSPC (9.4%); Choi (42.7%); ALC0159 (1.6%) Fll A016 (46.3%); DSPC (9.4%); Choi (42.7%); ALC0159 (1.6%) F12 A012 (46.3%); DSPC (9.4%); Choi (42.7%); ALC0159 (1.6%) F13 A003 (45%); DSPC (9%); Choi (44%); DMG-PEG2000 (2%) F14 A002 (41.67%); M4 (4.63%); DSPC (9.4%); CHOL (42.7%); DSPE PEG (1.6%)

[0567] F15 A001 (41.67%); M4 (4.63%); DSPC (9.4%); CHOL (42.7%); DSPE PEG (1.6%)

[0568] F16 A005 (41.67%); M4 (4.63%); DSPC (9.4%); CHOL (42.7%); DSPE PEG (1.6%)

[0569]

[0570] Control

[0571] LNP Composition

[0572] FC1 ALC-0315 (46.3%); DSPC (9.4%); Cholesterol (42.7%); ALCO 159

[0573] (1.6%)

[0574] FC2 ALC-0315 (41.67%); DOTAP(4.63%); DSPC (9.4%); Choi (42.7%);

[0575] DSPE-PEG2000 (1.6%)

[0576]

[0577] EXAMPLE 5

[0578] Comparative studies were performed by taking FC-2 (comparative formulation comprising ALC-0315 lipid) and F7 (comprising the inventive A003 lipid as per the present invention) LNPs. The characterization data (Average) of FC-2 and F-7 LNPs is tabulated below in TABLE 15:

[0579] TABLE 15:

[0580] LNP Average zeta size (nm) Average PDI Average EE% formulation

[0581] FC-2 145-174nm 0.135 42.37 (30-55%) F7 200-230nm 0.157 83.99 (80-88%)

[0582]

[0583] Adopted Methodology:

[0584] Preparation of LNPs

[0585] Lipid nanoparticles were prepared by the manual pipetting method. Lipids were dissolved in ethanol to prepare the stock solutions. Appropriate volumes from the lipid stock solution were taken to prepare the ethanolic lipid mixture in specific molar ratios (iLipid: cationic lipid: DSPC: Chol: PEG = 41.67: 4.63: 9.4: 42.7: 1.6). Forthe aqueous phase, Firefly luciferase mRNA (fLuc mRNA CleanCap 5-MoU; Trilink) at a specified concentration was added to sodium citrate buffer (35mM; pH 4). The ethanolic lipid mixture was rapidly mixed with the aqueous phase (with mRNA) in the ratio of 1:3 ethanol: aqueous (v / v) by manual pipetting and left undisturbed at room temperature for 15 minutes. The resulting formulation was diluted with 3 volumes of IX DPBS buffer containing 9% sucrose as a cryoprotectant, subjected to buffer exchange, and finally concentrated using Amicon Ultra-4 or 15 centrifugal filters (Merck Millipore). The final LNP formulation was cryostored by initially placing the vial at -20 °C for 1 hour, followed by transfer to -80 °C for long-term storage. The formulation was maintained on dry ice to preserve the required storage temperature and maintain the structural and functional integrity of the LNPs.

[0586] LNP Characterization

[0587] The size, polydispersity index (PDI), and zeta potential of the LNPs were determined by DLS measurements (Anton Paar Litesizer DLS 500, Austria). For this, a 10 pL LNP sample was diluted to 1 mL with lx DPBS (pH 7.4), and the sample size, PDI, and zeta potential were determined by DLS. The size distributions were calculated using a solvent refractive index of 1.33.

[0588] Example 6: Encapsulation efficiency

[0589] Encapsulation efficiency (EE%) of mRNA in LNPs were quantified using fluorescence platebased assay employing the Quant-iT™ Ribogreen® Assay Kit (Invitrogen) as per PNI Ribogreen assay protocol with some modifications

[0590]

[0591] user-guide-2020-final-emailsize.pdf). Briefly, mRNA standards (0-lpg / ml) were prepared by diluting mRNA stock (lOpg / ml) with lx TE buffer and 2% Triton-XlOO according to Table 16. For sample dilutions, LNPs were diluted 25-50 times in lx TE buffer so as to achieve mRNA concentrations in linear range of the standard curve. To measure the total RNA, 2% Triton-X100 was added to the diluted LNP samples and incubated at 70°C for 10 minutes. Ribogreen dye (lx, lOOpl) was added to each well and the plate was incubated for 5 minutes in dark at room temperature. Fluorescence readings were measured at 485nm / 528nm Ex / Em in SpectraMax M2e Plate reader.

[0592] Table 16: Preparation of mRNA standards for RNA standard curve

[0593] RNA cone. Vol (pl) from Vol of lx TE Vol of 2% Total vol (pl) lx Ribogreen (pg / ml) RNA stock (til) TX-100 (pl) dye (pl)

[0594] (lOpg / ml)

[0595] 1 20 30 50 100 100

[0596] 0.8 16 34 50 100 100

[0597] 0.6 12 38 50 100 100

[0598] 0.4 8 42 50 100 100

[0599] 0.2 4 46 50 100 100

[0600] 0 0 50 50 100 100

[0601]

[0602] Encapsulation efficiency was calculated as:

[0603] Total RNA — Unencapsulated RNA

[0604] EE% = X 100

[0605] Total RNA

[0606] Where, total RNA is measured from 2% Triton-XlOO treated well and unencapsulated RNA is measured from lx TE treated well.

[0607] Encapsulation efficiency and mRNA release were assessed for F7 (containing A003 and the novel cationic lipid, Lipid 1 from Patent 1 family), F14 (containing A002 and the novel cationic lipid, Lipid 1 from Patent 1 family), F15 (containing A001 and the novel cationic lipid, Lipid 1 from Patent 1 family), F16 (containing A005 and the novel cationic lipid, Lipid 1 from Patent 1 family) and FC2 (ALC-0315 + DOTAP). Ribogreen assay demonstrated that F7, F14, F15, F16 achieved an average encapsulation efficiency >68% significantly higher than -42.5% for FC2 (Figure 1). The improved mRNA release of the novel lipid-containing LNPs is mainly due to the pH-responsive linker in these lipids, which becomes protonated under mildly acidic conditions, allowing strong but controlled interactions with the mRNA cargo. The small amount of cationic lipid provides extra positive charge to support these interactions. This combination, along with the cone-shaped lipid geometry and helper phospholipids, promotes endosomal membrane destabilization and efficient intracellular release

[0608] The improved mRNA release of F7 is mainly due to the ionizable lipid A003. Its pH-responsive linker becomes protonated under mildly acidic conditions, allowing strong but controlled interactions with the mRNA cargo. The small amount of cationic lipid provides extra positive charge to support these interactions. This combination, along with the cone-shaped lipid geometry and helper phospholipids, promotes endosomal membrane destabilization and efficient intracellular release.

[0609] Table 17: Characterization data of FC2 and F7 LNPs

[0610] LNP Average PDI Average Encapsulation Efficiency%

[0611] FC2 0.135 42.37%

[0612] F7 0.157 83.99%

[0613] F14 0.183 68.44%

[0614] F15 0.206 70.85%

[0615]

[0616] F16 0.212 75.87%

[0617] Example 7 - Stability Data of LNPs, stored at 4°C

[0618] The LNPs of the present invention- F9, F5, F10, Fl and F12 containing 4-component (Ionizable lipids: DSPC: Chol: ALC0159) were stored at 4°C for 90 days to evaluate stability. Across all candidates, particle size, polydispersity index (PDI), and encapsulation efficiency (EE) remained stable for the full duration (Figure 2), demonstrating robust performance under standard refrigeration. This directly addresses a key limitation of many current mRNA-LNPs that require ultra cold storage, broadening their suitability for real world therapeutic settings. These ionizable lipids incorporate a pH responsive acetal oxolane linker that stabilizes lipid cargo interactions during circulation and cellular uptake. Controlled, gradual protonation under mildly acidic conditions supports endosomal fusion and escape while minimizing premature release. This contributes to overall nanoparticle integrity and functional stability during storage and use.

[0619] Replacing the ionizable lipid in a standard 4-component ALC-0315 based formulation with the ionizable lipids of the present invention preserved nanoparticle integrity during storage while maintaining size, PDI, encapsulation efficiency, and final cargo concentration. Extended stability at 4°C indicates potential for consistent performance across manufacturing lots, supports more flexible cold chain requirements, and enables smoother integration of these LNPs into established development and scale up workflows.

[0620] Example 8 - In vitro Transfection study

[0621] FC-2, F7, F14, F15, and F16 LNPs encapsulated with mCherry mRNA were used to transfect WT 9-7 cells (Patient-derived renal cysts cell lines) (ATCC, Virginia, USA) The WT 9-7 cells were seeded at a concentration of 50,000 cells / well in a 24-well plate and allowed to grow for 24 hours in the growth medium (DMEM supplemented with 10% fetal bovine serum (FBS) and penicillin (100 units / ml), streptomycin (100 pg / ml)). After 24 hours, the cells were washed with lx PBS, and mCherry encapsulated LNPs were subsequently delivered into cultured cells. Transfected cells were incubated at 37°C and 5% CO2. Lipofectamine MessengerMAX was used as an experimental standard / positive control for mRNA delivery. Transfection outcomes (cytoplasmic release of mcherry post uptake) were assessed by measuring mCherry expression and evaluating cell morphology at 6, 24 and 48 hours post transfection using EVOS M5000 microscope for fluorescence imaging and DIC microscopy.

[0622] The results revealed that the MessengerMax showing high transfection efficiency, serving as experimental control but shows significant cytotoxicity at 24 hours post-transfection. Although FC-2 showed better mRNA release in vitro when compared to F7, this advantage came at the cost of significant cytotoxicity, particularly in renal tissues.

[0623] The results revealed that the inventive formulations supported effective mCherry release at 6, 24 and 48 hours, confirming comparable intracellular payload delivery (Figure 4). Marked differences were observed in cytotoxicity: by 48 h, cells treated with FC2 exhibited severe morphological changes and extensive debris, whereas F7, F14, F15, F16 maintained reasonable cell integrity and viability even in these stressed, diseased kidney cells, demonstrating a favorable safety profile and potential utility in therapeutically relevant contexts (Figure 3a & 3b). The low cytotoxicity of F7 is largely attributed to the acetal oxolane family ionizable lipids. The gradual conversion of acetal rings to alcohols in these lipids produces largely non-reactive groups, minimizing nonspecific interactions with cell membranes and reducing liver uptake.

[0624] Example 9 - In vitro release and toxicity profiles of LNPs Fl, F2 & F3

[0625] A similar experiment, as described in example 7, was conducted using lipids Fl, F2, and F3 with the WT9-7 cell line. The effective release of mCherry mRNA was evaluated at 24 and 48 hours post-transfection. The release profiles of these formulations were comparable to the standard FC1 (ALC-0315) four-component LNP formulation. Notably, Fl and F2 exhibited toxicity profiles similar to FC1, whereas F3 demonstrated the lowest cytotoxicity among the tested formulations. A robust safety profile even in these stressed, diseased kidney cells, demonstrates the potential compatibility for therapeutic translation (Figure 4).

[0626] Example 10 - In vivo biodistribution study

[0627] Female mice model BALB / c aged 6-8 weeks (20-22gm) procured from IICT, Hyderabad) were used for in vivo biodistribution studies and maintained in cages under 12-h light / dark cycle at a controlled temperature (20 ± 2 °C) and relative humidity (50 ± 20%). They were given ad libitum to water and food and were allowed to acclimatise for 1 week before the experiments. All the animal experiments were done at IICT, Hyderabad according to the IEAC approved protocols.

[0628] One day prior to imaging, all the animals underwent removal of ventral abdominal hair from neck to tail for reducing the background signals. (Firefly luciferase) fLuc mRNA encapsulated LNPs (FC-2 LNPs and F7 LNPs) were administered to the mice via intravenous injections into the tail vein at a dosage of 0.5mg / kg fLuc mRNA and un-injected mice served as controls. Bioluminescence images for LNP biodistribution were taken after 6 hours of LNP injection. For in vivo imaging, animals were injected with D-luciferin (HY-12591B, MedChem Express) intraperitonially at a dose of 150mg / kg. After 5 minutes of D-luciferin injection, in vivo bioluminescence imaging was performed in IVIS system (IVIS Spectrum, Perkin Elmer). Animals were then sacrificed to isolate liver and kidneys for ex vivo imaging.

[0629] In vivo imaging showed abdominal fluorescence in the animals, suggesting abdominal accumulation of the LNPs. A good signal from the mice overall shows successful IV / tail vein injection i.e., the LNPs were effectively introduced into the bloodstream through tail vein injection, allowing for distribution throughout the body. Upon ex vivo imaging of liver and kidneys, both the formulations showed signals indicating the biodistribution of the LNP formulations to liver and kidney. This can be seen from the images in Figure 5.

[0630] However, FC-2 showed approximately 11 -fold higher signal in liver as compared to F7 formulation. The p value < 0.05 in case of liver demonstrates that the difference in biodistribution between the formulations is statistically significant. F7 exhibited comparable kidney biodistribution with far less off-target accumulation in the liver and minimal renal toxicity. The reduced liver tropism seen with F7, combined with its consistency across batches and lower cytotoxicity, highlights its potential as a safer and more reliable candidate for kidney-targeted therapies. These results suggest that while FC-2 offers improved release in vitro, its cytotoxicity limits its utility, making F7 the more suitable formulation for future development in mRNA therapeutics targeting the kidney.

[0631] Apart from achiveing at least a 11-fold detargetting of the liver, the formulations comprising the LNPs of the present invention have further demonstrated to have significantly lower toxicity. The low toxicity profile displayed by F7 in comparison to FC-2 can also be attributed to the presence of the novel lipid A003 in F7. The design of A003 enables better biodegradability of the formulation into water soluble particles immediately post release of the cargo leading to minimal toxicity and damage.

[0632] The kidney tropism of F7 is driven by its ionizable lipid, whose acetal-oxolane linker undergoes gradual protonation under mildly acidic conditions, converting into hydroxyl groups that slow degradation and reduce hepatic uptake. This pH-responsive stabilization prolongs systemic circulation. The small amount of cationic lipid provides complementary stabilization with similar resistance to rapid endosomal degradation, and together these lipids fine-tune surface charge to favor renal rather than liver targeting. F7 exhibits an optimal combination of size, charge, and pKa necessary for kidney uptake, which differs from the conditions for hepatic uptake.

[0633] Example 11

[0634] A similar set of experiment a described in example 10 was conducted with LNPs F4, F5, F6 and control FC1.

[0635] The results revealed that the standard formulation FC1 showed predominantly hepatic delivery and baseline cytotoxicity.

[0636] In F4, the PEG lipid was modified to DSPE-PEG2000 for further enhancing extrahepatic delivery, but tuning the outer-shell PEG alone showed only minimal effect. F5 showed a 3-4-fold increase in extrahepatic delivery (~1.0 x 107) while maintaining low cytotoxicity, highlighting the superior performance of acetal oxolane family ionizable lipids. In F6, the introduction of a fifth lipid component (A003 + Addition of specific cationic Lipid ~4 mol%) and replacement of PEG-ALC0159 with an optimized PEG drastically reduced liver accumulation (~1.0 x 105). This demonstrates that careful combination of acetal oxolane family lipids with other lipid components can maximize extrahepatic delivery and minimize hepatic uptake of the formulation (Figures 6a, 6b and 6c).

[0637] Example 12 - Tissue toxicity study

[0638] Renal artery (RA) injections were performed following IEAC-approved protocols. Mice were anesthetized with a ketamine-xylazine cocktail, adjusted for body weight. An abdominal incision exposed the left kidney, and the renal artery was carefully isolated. LNP formulations (FC-2 and F7) were injected slowly into the artery using an insulin syringe. The injection site was sealed with bio adhesive, and the incision was closed with sutures and treated with povidone-iodine. Post-surgery, animals were monitored in warm conditions and given antibiotics. After 24 hours, mice were sacrificed, and both kidneys were harvested for toxicity analysis. Tissues underwent fixation in 4% PFA, followed by sucrose gradient treatment. Sagittal sections of frozen samples were prepared for bright field imaging to assess tissue toxicity.

[0639] Toxicity analysis for FC-2 and F7 LNPs was done by comparing their DIC images of cryosections. FC-2 LNP was found to be more cytotoxic than F7 LNP for the cortical and the medullary region of the kidney as revealed by DIC images.

[0640] Fig. 7 reflects the comparison of DIC images of kidney sections of un injected, FC-2 and F7 LNPs, after 24 hours of renal artery injection observed through bright field microscopy. Dead cells / debris denoting cytotoxicity of FC_2 LNP can be seen in the FC-2 injected kidney. FC-2 was found to be more cytotoxic than F7 by comparing the cell morphology, observation of more rounded cells and more cell debris in FC-2, which signify higher cytotoxicity. This can be attributed to the poor encapsulation efficiency of FC-2 leading to excessive use of lipids to encapsulate the same amount of mRNA when compared to F7. Figure7 reflects the lower cytotoxicity of the F7 formulation as compared to the FC-2 formulation.

[0641] Further studies focus on two major aspects: a. LNP delivery at the organ level in the heavily cystic PKD1 knock out mouse model, Pkdl-flox and Cdhl6-CreERT2 (Pkdlfl / flCdhl6CreERT2), and

[0642] b. LNP delivery to specific target cell types i.e, Proximal Tubular Epithelial Cells (PTECs) and Collecting Ducts (CDs) within this model.

[0643] Example 13- Breeding and strain validation of Pkdl-flox and Cdhl6-CreERT2 mice (heavily cystic PKD1 mouse model)

[0644] C57BL / 6JGpt-PkdlemlCflox / Gpt (No. T008136, hereafter referred as Pkdl-flox) and C57BL / 6JGpt-Cdhl6emlCin (CreERT2) / Gpt (No. T007046, hereafter referred as Cdhl6-CreERT2) strains were used for the breeding and generation of Pkdlfl / flCdhl6CreERT2 mice model at GemPharmatech (GPT), China. The mouse model were procured from CRO partner Gem Pharmatech, China. Induction of PKD1 -associated cyst formation was performed by intraperitoneal (IP) injection of tamoxifen at a dose of 100 mg / kg / day in 3 -week-old mice. The model was validated for cyst development using multiple evaluation modalities, including kidney ultrasound, assessment of kidney morphology ex vivo, measurement of serum BUN and creatinine levels, calculation of the kidney index, and histopathological examination of kidney tissues through H& E staining.

[0645] The mice exhibited pronounced kidney cyst formation upon morphological examination. Detailed histopathological analysis revealed significant renal alterations in the model, including multiple cysts, abundant protein casts, tubular atrophy, and extensive interstitial fibrosis. Quantitatively, these mice demonstrated a marked increase in kidney index compared to controls. Biochemical assessment showed significantly elevated serum blood urea nitrogen (BUN) and creatinine (CREA) levels, which progressively worsened with age, consistent with declining renal function.

[0646] Additionally, ultrasound imaging serves as a non-invasive and reliable tool for monitoring kidney volume changes in PKD1 cystic mice. Measurement of total kidney volume through ultrasound provides a quantitative indicator of cyst burden and correlates strongly with functional decline, making it an essential parameter for validating disease severity and therapeutic efficacy in preclinical PKD1 studies. Further validation of the PKD1 strain was carried out using ultrasound imaging at 13 weeks which revealed enlarged kidney volumes of around 700-1500mm3 (Table 5) as compared to the kidney volume of healthy mice which is approximately 500-600mm3 at 12-13 weeks of agel2,13, further corroborating disease progression. Table 18. Size as well as volume measurement of Left and Right kidneys of Pkdlfl / flCdhl6CreERT2 mice at around 13 weeks of age through ultrasound

[0647]

[0648] The observed cystic transformation and associated pathological features in Pkdl:fl / fl Cdhl6-CreERT2:ki / wt mice closely recapitulate the phenotypic spectrum of autosomal dominant polycystic kidney disease (ADPKD). The presence of multiple cysts, tubular atrophy, and increased fibrotic deposition highlight the model’s suitability for studying cystogenesis and progressive renal damage. The significant rise in kidney index and progressive elevation of serum BUN and creatinine levels indicate a functional decline parallel to histological damage. Additionally, increased kidney volumes detected by ultrasound further support the development and enlargement of cysts over time in this model. Collectively, these results establish the Pkdl:fl / fl Cdhl6-CreERT2:ki / wt mouse as a robust preclinical tool for investigating ADPKD pathogenesis and evaluating potential therapeutic interventions.

[0649] Example 14- F7 amenable to local routes of administration (retrograde ureteral and renal artery injections)

[0650] Retrograde Ureteral Injection: Administration of F7 via the retrograde ureteral (RU) route resulted in highly localized kidney accumulation, with negligible liver distribution. In vivo and ex vivo imaging at 6 h post-injection confirmed strong renal tropism, thereby validating the utility of F7 for localized kidney delivery (Figure 8). This route -dependent specificity highlights not only the targetability of the F7 LNP, but also its robustness in withstanding direct injection into a high-pressure organ environment such as the kidney. The ability to maintain encapsulation, avoid cytotoxicity, and demonstrate controlled release under such physiologically demanding conditions underscores its translational potential for one-time or infrequently redosed therapies such as cell and gene therapies.

[0651] Renal artery injection: Renal artery administration of F7 resulted in markedly enhanced kidney accumulation relative to intravenous dosing, with distribution largely confined to renal tissue. Notably, the particles withstood the elevated shear and pressure conditions of renal arterial flow, underscoring the formulation’s structural stability (Figure 9).

[0652] Example 15 - Biodistribution of F7 in Disease Mice Models

[0653] In the PKD1 knockout model (Pkdlfl / fl; Cdh 16-CreFRT2ki''') of ADPKD, F7 was administered at 12 weeks of age, when kidneys are highly cystic and the disease phenotype is advanced. Both intravenous (IV) and retrograde ureteral (RU) injections were performed, and organ-level biodistribution was assessed 6 h post-administration. In both routes, kidney delivery was observed, with RU yielding markedly higher renal accumulation. These findings confirm that F7 retains its kidney-targeting capacity even under conditions of severe structural distortion (enlarged and heavily cystic kidneys), underscoring its translational relevance for treating advanced kidney disease (Figure 10).

[0654] Example 16 - Cell-specific Uptake in Disease Mice Models Using F8

[0655] Cell-specificity analysis of F8 (0.4 mg / kg) administered via the retrograde ureteral route in the Pkdlfl / fl; Cdhl6-CreERT2ki / wt model at 13-14 weeks of age demonstrated homogenous renal epithelial uptake. Immunofluorescence at 6 h post-injection revealed strong reporter expression in both collecting duct (AQP2) and proximal tubular (URP2) epithelial cells (Figure 11). Mice were sacrificed, and perfusion with saline was performed, followed by tissue collection. The kidneys were all segmented along the transverse section, fixed and paraffin embedded. Four sections were cut from each kidney paraffin block (3-5pm thickness): one section served as a no antibody control, the second section was treated with only fLuc antibody, the third section was treated with antibodies against fLuc (Abeam) and proximal tubule (megalin; LRP2 Santa Cruz), and the fourth section was treated with antibodies against fLuc and collecting duct (aquaporin2; AQP2 Santa Cruz). The dilution ratios used were fLuc (1:200), AQP2 (1:500), and LRP2 (1:500). Antigen retrieval was performed using Tri-EDTA high-temperature heat retrieval for 40 minutes.

[0656] After staining, the images were acquired according to the following parameters:

[0657] IF staining-image Acquisition Parameters

[0658] Index Exposure time

[0659] Single-label fLuc (Cy5) Cy5-8ms

[0660] Double-label AQP2(FITC)+Fluc (Cy5) FITC-30ms, Cy5-8ms LRP2(FITC)+Fluc (Cy5) FITC-30ms, Cy5-8ms

[0661]

[0662] Co-localization analysis was carried out using Image J software on the acquired images. Colocalization analysis of the reporter protein (fLuc) with collecting ducts (marked by AQP2) and proximal tubular cells (marked by LRP2) showed a positive spatial correlation as indicated by Pearson's coefficient. When Manders' coefficient was calculated, it was found that significant proportions of total AQP2 signal and LRP2 signal overlapped with the reporter protein. Remaining signal from cell specific markers co-existed with the signal from the reporter protein indicating that nearly all the labelled cells had taken up and expressed the cargo, but only a fraction of the signal showed co-localization with the reporter protein. Furthermore, Costes' randomization test yielded a p-value of 100% in both cases, indicating that the observed colocalization was genuine, highly significant, and not due to random chance.

[0663] The optimized lipid composition in F8 enhances targeting tubular epithelial cells and also enables delivery to cyst lining cells and cysts themselves. The unique composition of F8 displays an optimal size, charge, and pKa that allow for longer interaction of the LNPs with the tubular epithelial cells even in the scenario of the retrograde ureteral injection, which is against the flow of urine and hence can lead to washing away of the LNPs before uptake by the target cells. This cell-specific uptake underscores the therapeutic relevance of F8 for kidney diseases rooted in renal tubular epithelial dysfunction.

[0664] Example 17 - In vivo Tolerability of F7 Retrograde ureteral (RU) injection: BALB / c mice were administered F7 via the RU route, and kidneys were collected 24 h later for histopathological analysis. Examination of both major organs and the injected kidney revealed no evidence of moderate to severe tissue damage (Figure 12). This favorable safety profile aligns with the preserved cell viability observed in vitro, confirming the biocompatibility of the optimized formulation under physiologically demanding conditions.

[0665] Renal artery (RA) injection: Tissue toxicity was assessed 24 h post-renal artery administration. Differential interference contrast (DIC) imaging of cryosections showed that FC2 (ALC-0315 + DOTAP) induced notable cytotoxicity in the medullary region, whereas F7 maintained tissue integrity with minimal adverse changes (Figure 13).

[0666] Together, these findings extend the earlier in vitro observations by confirming that the F7 architecture reduces membrane-disruptive toxicity in vivo. In vivo studies show that F7, formulated with A003 ionizable lipids, exhibits lower toxicity compared with ALC-0315 based formulations. While ALC-0315 lipid can generate reactive byproducts upon degradation, A003 features a pH-responsive acetal-oxolane linker that is gradually protonated under mildly acidic conditions, slowly converting to hydroxyl groups. This controlled degradation enhances fusogenic properties, promotes efficient endosomal escape, and supports effective intracellular mRNA release, all while minimizing cytotoxic effects.

[0667] Example 18 - Effective Genome Editing in Kidney Cell Line in TKPTS Using F13 LNP (4C with A003)

[0668] Functional validation of delivery was confirmed through T7E1 endonuclease assay measuring gene editing experiments in the TKPTS cell line, an immortalized mouse proximal tubular epithelial cell model. F13 LNPs containing the A003 ionizable lipid enabled efficient codelivery of CRISPR-Cas9 mRNA and mouse PKD1 single guide RNA, with editing efficiency at 48 hours comparable to ALC-0315-based FC3 LNPs, while FC4 LNPs containing MC3 did not display any detectable editing. Taken together, these results demonstrate that F 13 supports functional genome modification, extending its utility beyond mRNA payload delivery to therapeutic genome editing applications. Genomic DNA was isolated from TKPTS cells using the DNeasy Blood & Tissue Kit (Qiagen, 50-prep format) according to the manufacturer’s protocol. Briefly, cell pellets were lysed in Buffer ATL and Proteinase K, followed by ethanol addition and binding to the silica membrane column, with subsequent wash steps using Buffers AW1 and AW2 before final elution in nuclease-free water. Target-specific DNA regions, including CRISPR editing sites, were amplified by PCR using PrimeSTAR GXL DNA Polymerase (Takara) according to the recommended cycling conditions for high-fidelity amplification of genomic templates. PCR products were then subjected to mismatch cleavage analysis using the Alt-R™ Genome Editing Detection Kit (Integrated DNA Technologies). In accordance with the manufacturer’s instructions, equal amounts of amplicons were denatured and re-annealed to form heteroduplexes, digested with the supplied T7 Endonuclease I, and analysed by agarose gel electrophoresis to assess genome editing efficiency (Figure 14).

Claims

1. Claims:

1. A lipid of Formula I or its pharmaceutically acceptable salt, tautomer, prodrug or stereoisomer thereof with the following structure:

4. 6.wherein R5 is7.hydrogen; C1-C3 alkyl; C3-C7 cycloalkyl; aryl, heteroaryl or heterocycloalkyl; -(CH2)OC(R)2(CH2)0-18-OQ, -C(O)NQR or -(CH2)0-18Q, in which Q is H, OH, -NHC(S)N(R)2, -NHC(O)N(R)2, -N(R)C(O)R, -N(R)S(O)2R, -N(R)Rc. -NHC(=NR)N(Rc). - NHC(=CHR)N(Rc)2. -OC(O)N(R)2, -N(R)C(O)OR¢; wherein R and Rc are same or different and are selected from hydrogen; C1-C3 alkyl; C3-C7 cycloalkyl; aryl, heteroaryl or heterocycloalkyl, or a group selected from:

9.

10. 11.

13.

14. R4Wherein the ‘*’ denotes the point of attachment to the ‘G’ group.15.G is a single, double or triple bond; unsubstituted or substituted C1-C3alkyl, -(CH2)0-18-; -O(C=O)-; -O(C=O)O; -O(C=S)O; S-S; -O-; -(C=O)S(CH2)0-18; -NH(C=O)-;16.Z is either C orN;17.Xi, X2, X3 and X4 are selected from C, NH, O, or S provided at least two of Xi, X2, X3 and X4 are O; A i and A2 are each independently C 1 -C3 alkylene group; A 1 and A2 can optionally together with the Z-atom to which they are attached form a 5 membered ring;18.Li and L2 are the same or different and are each independently selected from19.substituted or unsubstituted C1-C12 alkylene or C1-C12 alkenylene;20.-C(O)O-, -OC(O)-, -C(O)N(R )-, -P(O)(OJ )O-;21.(CH2)O-I8-; -OC(0)(CH2)O-I8-; -OC(O)O(CH2)0-i8-; -CH(OR )2-; -OCOCH(R )2-;22.-C(O)O(CH2)0-18-;23.-C(O)O(CH2)0-18(C2H2)0-18CH-;24.-C(O)OCH-;25.where Rcc is hydrogen and un / substituted C1-3 alkyl; -S-S-, an aryl group, and a heteroaryl group;26.Ri, R2 are independently selected from the group consisting of hydrogen, straight chain or branched27.Ci-Cis alkyl, Ci-Cis alkenyl, Ci-Cis alkynyl;28.C0-C18 alkyl- D - Ci-Cis alkyl;29.C0-C14 alkylene- D - Ci-Cis alkylene;30.C0-C18 alkyl- D - Ci-Cis alkylene;31.C0-C14 alkylene- D - Ci-Cis alkyl;32.where D is -C(O)-; -COO-; -C(O)S-; -O-; -S-;33.and further34.Ri and R2 optionally together with the carbon atom to which they are attached form a 3-5 membered heterocyclic ring with RI and R2 being selected from O or S; wherein the 3-5 membered heterocycle ring is optionally substituted with straight chain or branched Ci-Cis alkyl, C1-C14 alkenyl, C0-C14 alkyl- D - Ci-Cis alkyl; C0-C14 alkylene- D - Ci-Cis alkylene;, C0-C14 alkyl- D - Ci-Cis alkylene;, C0-C14 alkylene- D - Ci-Cis alkyl; where D is -C(O)-; -COO-; -C(O)S-; -O-; -S-;35.R3 and R4 are independently selected from the group consisting of H, straight chain or branched Ci-Cis alkyl, C1-C14 alkenyl, C0-C14 alkyl- D - Ci-Cis alkyl; C0-C14 alkylene- D -Ci-Cis alkylene;, C0-C14 alkyl-D- Ci-Cis alkylene;, C0-C14 alkylene-D- Ci-Cis alkyl; where D is -C(O)-; -COO-; -C(O)S-; -O-; -S-; and36.m is 0-9; p is 0-9; b is 0-12; c is 0-12; n is 0-9, and n’ is 0-9.

2. The lipid as claimed in claim 1, having a Formula 1-A38. 39.R4 Formula 1-A40.wherein41.Ai and A2 are each independently C1-C3 alkylene group; Re and R7 are independently selected from straight chain or branched Ci-Cis alkyl, C1-C14 alkenyl, C0-C14 alkyl- D - Ci-Cis alkyl; C0-C14 alkylene- D - Ci-Cis alkylene; C0-C14 alkyl- D - Ci-Cis alkylene; C0-C14 alkylene- D -Ci-Cis alkyl; where D is -C(O)-; -COO-; -C(O)S-; -O-; -S-;42.and wherein R3, R4, R5, m, p, b, c are as defined for formula (I).

3. The lipid as claimed in claim 1, having a Formula 1-B45. 47.Wherein48.Ai and A2 are each independently C1-C3 alkylene group;49.Re and R7 are independently selected from straight chain or branched Ci-Cis alkyl, C1-C14 alkenyl, C0-C14 alkyl- D - Ci-Cis alkyl; C0-C14 alkylene- D - Ci-Cis alkylene;, C0-C14 alkyl- D - Ci-Cis alkylene;, C0-C14 alkylene- D - Ci-Cis alkyl; where D is -C(O)-; -COO-; -C(O)S-; -O-; -S-; R3, R4, Rs, m, p, b, c are as defined for formula (I).

4. The lipid as claimed in claim 1, having a Formula 1-C51.

52. Formula 1-C53.Wherein54.Ai and A2 are each independently C1-C3 alkylene group;55.Re and R7 are independently selected from straight chain or branched Ci-Cis alkyl, C1-C14 alkenyl, C0-C14 alkyl- D - Ci-Cis alkyl; C0-C14 alkylene- D - Ci-Cis alkylene;, C0-C14 alkyl- D - Ci-Cis alkylene;, C0-C14 alkylene- D - Ci-Cis alkyl; where D is -C(O)-; -COO-; -C(O)S-; -O-; -S-; R3, R4, Rs, m, p, b, c are as defined in formula (I).

5. The lipid as claimed in claim 1, having a Formula 1-D58.

59. Formula 1-D60.Wherein Ai and A2 are each independently C1-C3 alkylene group;61.Re and R7 are independently selected from straight chain or branched Ci-Cis alkyl, C1-C14 alkenyl, C0-C14 alkyl- D - Ci-Cis alkyl; C0-C14 alkylene- D - Ci-Cis alkylene;, C0-C14 alkyl- D - Ci-Cis alkylene;, C0-C14 alkylene- D - Ci-Cis alkyl; where D is -C(O)-; -COO-; -C(O)S-; -O-; -S-; R3, R4, Rs, m, p, b, c are as defined in formula (I).

6. The lipid as claimed in claim 1, having a Formula 1-E63. 65.Formula 1-E66.Wherein Ai and A2 are each independently C1-C3 alkylene group; R3, R4, Rs, m, p, b, c are as defined in formula (I).

7. The lipid as claimed in claim 1, having a Formula 1-F69.

70. R471.Formula 1-F72.wherein73.Ai and A2 are each independently C1-C3 alkylene group; R3, R4, Rs, m, p, b, c are as defined in formula (I).

8. The lipid as claimed in claim 1, having a Formula 1-G75. 77.Formula 1-G78.Wherein79.Ri and R2 are each independently hydrogen, Ci-Cis alkyl, Ci-Cis alkenyl, or Ci-Cis alkynyl; R3, R4, R5, m, p, b, c are as defined in formula (I).

9. The lipid as claimed in claim 1, having a Formula 1-H82. 84.Formula 1-H85.Wherein86.Ai and A2 are each independently C1-C3 alkylene group; Re and R7 are independently selected from straight chain or branched Ci-Cis alkyl, C1-C14 alkenyl, C0-C14 alkyl- D - Ci-Cis alkyl; C0-C14 alkylene- D - Ci-Cis alkylene;, C0-C14 alkyl- D - Ci-Cis alkylene;, C0-C14 alkylene- D - Ci-Cis alkyl; where D is -C(O)-; -COO-; -C(O)S-; -O-; -S-; R3, R4, R5, m, p are as defined in formula (I).

10. The lipid as claimed in claim 1, having a Formula 1-188.

89. R490.Formula 1-191.Wherein92.Ai and A2 are each independently C1-C3 alkylene group;93.Re and R7 are independently selected from straight chain or branched Ci-Cis alkyl, C1-C14 alkenyl, C0-C14 alkyl- D - Ci-Cis alkyl; C0-C14 alkylene- D - Ci-Cis alkylene;, C0-C14 alkyl- D - Ci-Cis alkylene;, C0-C14 alkylene- D - Ci-Cis alkyl; where D is -C(O)-; -COO-; -C(O)S-; -O-; -S-; and wherein R3, R4, R5, m and p are as defined in formula (I).

11. The lipid as claimed in claim 1, having a Formula 1-J96. 98.Formula 1-J99.Wherein100.Ai and A2 are each independently C1-C3 alkylene group;101.R6 and R7 are independently selected from straight chain or branched Ci-Cis alkyl, C1-C14 alkenyl, C0-C14 alkyl- D - Ci-Cis alkyl; C0-C14 alkylene- D - Ci-Cis alkylene;, C0-C14 alkyl- D - Ci-Cis alkylene;, C0-C14 alkylene- D - Ci-Cis alkyl; where D is -C(O)-; -COO-; -C(O)S-; -O-; -S-; R3, R4, R5, m, p are as defined in formula (I).

12. The lipid as claimed in claim 1, having a Formula 1-K104.

105. 0106.Formula 1-K107.wherein Ai and A2 are each independently C1-C3 alkylene group; Ri and R2 are each independently hydrogen, Ci-Cis alkyl, Ci-Cis alkenyl, or Ci-Cis alkynyl; or Ri and R2 optionally together with the carbon atom to which they are attached form a 5 membered heterocyclic ring with RI and R2 being selected from O; wherein the 5 membered heterocycle ring is optionally substituted with straight chain or branched Ci-Cis alkyl, C1-C14 alkenyl, Co-C14 alkyl- D - Ci-Cis alkyl; C0-C14 alkylene- D - Ci-Cis alkylene;, C0-C14 alkyl- D - Ci-Cis alkylene;, C0-C14 alkylene- D - Ci-Cis alkyl; where D is -C(O)-; -COO-; -C(O)S-; -O-; -S-; and wherein R3, R4, R5, m, b, c are as defined in formula (I).

13. The lipid as claimed in claim 1, having a Formula 1-L109.0110.Formula 1-L112.

113. wherein Ai and A2 are each independently C1-C3 alkylene group; Ri and R2 are each independently hydrogen, Ci-Cis alkyl, Ci-Cis alkenyl, or Ci-Cis alkynyl; or Ri and R2 optionally together with the carbon atom to which they are attached form a 5 membered heterocyclic ring with RI and R2 being selected from O; wherein the 5 membered heterocycle ring is optionally substituted with straight chain or branched Ci-Cis alkyl, C1-C14 alkenyl, Co-C14 alkyl- D - Ci-Cis alkyl; C0-C14 alkylene- D - Ci-Cis alkylene;, C0-C14 alkyl- D - Ci-Cis alkylene;, C0-C14 alkylene- D - Ci-Cis alkyl; where D is -C(O)-; -COO-; -C(O)S-; -O-; -S-; and wherein R3, R4, R5, m, b, c are as defined in formula (I).

14. The lipid as claimed in claim 1, having a Formula 1-M115.H116.N118. 119.4Formula 1-M120.wherein Ai and A2 are each independently C1-C3 alkylene group;121.and wherein R3, R4, R5, m, p, b, and c are as defined in formula (I).

15. The ionizable lipid of formula (I) as claimed in claim 1, wherein the compound is123.H3CXH3C125.

127.

128. p130.

16. A lipid formulation comprising at least one ionizable lipid of formula (I) as claimed in any one of the preceding claims, a cholesterol, one or more of PEGylated lipid or / and helper lipid.

17. The formulation as claimed in claim 16, comprising 30-70% by weight of at least one ionizable lipid of formula I, 10-30 % by weight of cholesterol, 15-30 % by weight of one or more PEGylated lipid or / and helper lipid.

18. The formulation as claimed in claim 16, comprising 30-60% by weight of at least one ionizable lipid of formula I, 35-50% by weight of cholesterol, 1-5% by weight of PEGylated lipid, and 7-10% by weight of helper lipid.

19. The formulation as claimed in claim 16, wherein the formulation comprises cationic lipid.

20. The formulation as claimed in claim 16, comprising 30-50% by weight of at least one ionizable lipid of formula I, 1-10 % by weight of cationic lipid, 35-45% by weight of cholesterol, 1-10% by weight of PEGylated lipid and 5-15% by weight of helper lipid.

21. The lipid formulation as claimed in claim 16, wherein ionizable lipid: cationic lipid: lipid:DSPC:Chol:PEG are present in the ratio 41.67: 4.63: 9.4: 42.7: 1.6.

22. The lipid formulation comprising at least one lipid of Formula I as claimed in claim 16, wherein the formulation encapsulates a therapeutic agent for target cell delivery beyond liver with delivery to other organs such as but not limited to kidney, heart, spleen, lung, lymphatic tissue.

23. The lipid formulation comprising at least one lipid of Formula I as claimed in claim 16, wherein the formulation encapsulates one or more therapeutic agents for target cell delivery to a diseased organ including but not limited the kidney, liver, lung, pancreas, or any other organs and target cell types including but not limited to epithelial, endothelial, stromal, and other specialized cell types, including kidney-associated proximal convoluted tubule, distal convoluted tubule, and collecting duct cells.

24. The lipid formulation comprising at least one lipid of Formula I as claimed in claim 16, wherein the formulation encapsulates a therapeutic agent for target cell delivery with selective cystic or fibrotic kidney tropism.

25. A method of delivering a payload comprising encapsulating a therapeutic agent in the lipid formulation as claimed in claim 16 and administering the lipid formulation comprising the therapeutic agent into the body of a subject.

26. The method as claimed in claim 25, wherein the lipid formulation is administered through intravenous, intra-arterial, intraperitoneal, intraparenchymal, subcutaneous, intramuscular, intradermal, intrathecal, intraocular, inhalational, intranasal, buccal, sublingual,oral, transdermal, intrapulmonary, intralymphatic, intranodal, intratumoral, intracavitary, intratracheal, or catheter-based, device-assisted, or image-guided administration.

27. The method as claimed in claim 25, wherein the lipid formulation is administered through intravenous (IV) route, renal artery injection, retrograde ureteral injection (RU), renal vein injection, peritoneal injection, subcapsular injection, intra-parenchymal injection to reach different cell types within the kidney.

28. The method as claimed in claim 25 wherein the therapeutic agent is a nucleic acid-based material such as RNA (including mRNA, saRNA, siRNA, shRNA, miRNA, and guide RNAs), DNA, anti-sense oligonucleotides, plasmids, gene-editing components, and nucleic acid analogs; proteins and peptides including enzymes, antibodies, antibody fragments, cytokines, growth factors, and peptide therapeutics; small molecules of natural, synthetic, or semi-synthetic origin; immunomodulatory agents including vaccines, adjuvants, immune stimulants, or immune suppressants; biologies such as viral vectors, fusion proteins, aptamers, and polysaccharides; diagnostic or imaging agents including contrast agents and reporter constructs; and prodrugs or precursor molecules capable of conversion to an active form in vivo.

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